Language：Japanese   Publishing type：Research paper (scientific journal)   Participation form：Joint(The vice charge)  

Language：Japanese   Publishing type：Research paper (other academic)   Participation form：Joint(The main charge)  

Language：Japanese   Publishing type：Research paper (scientific journal)   Participation form：Joint(The vice charge)  

Language：Japanese   Publishing type：Research paper (scientific journal)   Participation form：Joint(The vice charge)  

Language：Japanese   Publishing type：Research paper (scientific journal)   Participation form：Joint(The main charge)  

Language：English   Publishing type：Research paper (scientific journal)   Participation form：Joint(The vice charge)  

DOI： 10.1007/s10967-018-6304-4

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Plant Cell and Environment  

© 2017 John Wiley & Sons Ltd Many aquatic plants act as biosorbents, removing and recovering metals from the environment. To assess the biosorbent activity of Egeria densa, a submerged freshwater macrophyte, plants were collected monthly from a circular drainage area in Lake Biwa basin and the Mn concentrations of the plants were analysed. Mn concentrations in these plants were generally above those of terrestrial hyperaccumulators, and were markedly higher in spring and summer than in autumn. Mn concentrations were much lower in plants incubated in hydroponic medium at various pH levels with and without Mn supplementation than in field-collected plants. The precipitation of Mn oxides on the leaves was determined by variable pressure scanning electron microscopy-energy dispersive X-ray analysis and Leucoberbelin blue staining. Several strains of epiphytic bacteria were isolated from the field-collected E. densa plants, with many of these strains, including those of the genera Acidovorax, Comamonas, Pseudomonas and Rhizobium, found to have Mn-oxidizing activity. High Mn concentrations in E. densa were mediated by the production of biogenic Mn oxide in biofilms on leaf surfaces. These findings provide new insights into plant epidermal bacterial flora that affect metal accumulation in plants and suggest that these aquatic plants may have use in Mn phytomining.

DOI： 10.1111/pce.12910

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Language：English   Publishing type：Research paper (scientific journal)   Participation form：Joint(The main charge)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Metallomics: Recent Analytical Techniques and Applications  

© Springer Japan KK 2017. X-ray microanalyses have been utilized to determine mechanisms of metal detoxification and homeostasis in plants and are powerful tools, especially for target plants unsuitable for genetic analyses. This chapter describes several synchrotron-based research methods to determine metal element distribution and speciation of metal-accumulating plants. The procedures are described, to show how micro-X-ray techniques are applicable to various biological materials, including those that are solid or liquid, soft or hard, or wet or dry. Combination of X-ray microanalysis with other methodologies, including TEM (transmission electron microscope), SEM (scanning electron microscope), and techniques of biochemistry and molecular biology, is described. Overall, this chapter attempts to encourage cross-disciplinary work in metallomics.

DOI： 10.1007/978-4-431-56463-8_6

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Language：Japanese   Publishing type：Research paper (scientific journal)   Participation form：Joint(The main charge)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society for Plant Cell and Molecular Biology   Participation form：Joint(The main charge)  

DOI： 10.5511/plantbiotechnology.15.1214a

Language：Japanese   Publishing type：Research paper (conference, symposium, etc.)   Participation form：Joint(The main charge)  

Language：English   Publishing type：Research paper (other academic)   Participation form：Joint(The main charge)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：CSIRO  

DOI： 10.1071/BT14242

Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Analytical Atomic Spectrometry  

Radioactive cesium (Cs) was discharged into the environment in the northeast region of Japan after destruction of the nuclear power station in March 2011. In this work, we study the uptake and accumulation of Cs in a freshly submerged vascular plant, Egeria densa (Brazilian waterweed, Hydrocharitaceae), to investigate the behavior of Cs in the aquatic ecosystem and to predict the movement of the radionuclide to remove this Cs from the environment. We collected plants, waters, and sediments that had been contaminated by radioactive fallout in Fukushima Prefecture in September 2012. The activities of 134Cs, 137Cs, and 40K were measured using a germanium semiconductor detector. We found that the endogenous radioactive Cs concentrations in plants were correlated with those in the sediments. To discuss the uptake mechanism of Cs, stable 133Cs was applied separately to shoots and roots in a two-compartment bath. The results showed that the environmental Cs transferred to both shoot and root parts in plants. To analyze the detailed localization of Cs in plants, synchrotron radiation-based micro X-ray fluorescence (SR-μ-XRF) analysis was performed for the plants cultivated in a hydroponic medium containing 133Cs. The two-dimensional metal distribution of leaves was determined by μ-XRF with approximately 0.7 μm X-ray beams. The distribution showed the predominant localization of Cs, K as well as several divalent metals in the cell wall or apoplastic regions, suggesting the possible absorption mechanism of Cs in the plants. We concluded that E. densa plays an important role in the deposition of radioactive Cs in fresh water. © 2014 The Partner Organisations.

DOI： 10.1039/c3ja50346a

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Metallomics  

Trees that accumulate metals are important plants for restoring contaminated soil because of their high biomass. In our previous study, we discovered that Salix miyabeana has the capability to take up high levels of Cd, and identified the several accumulation sites of the endogenous metals in the leaf parts of plants. To analyze the detailed localization of Cd in apoplastic and symplastic compartments in S. miyabeana, synchrotron radiation-based micro X-ray fluorescence (SR-μ-XRF) analysis and micro X-ray absorption near edge structure (μ-XANES) measurements were performed on beam line 37XU of the SPring-8 (Hyogo, Japan). The two-dimensional metal distribution of segments of young stems was obtained by μ-XRF with approximately 2 μm 2 X-ray beams and showed the predominant localization of Cd in the apoplastic region. μ-XANES analyses suggest that the apoplastic detoxification of Cd in willow depends on Cd-oxygen, but not on Cd-sulfur, interaction. S. miyabeana growing near an old mining site in Japan was then examined to evaluate the metal accumulating ability of this plant in the field. The metal concentration in the leaves of the plants was compared to that in the soil and enrichment factors (EFs) were calculated for Cu, Zn, Cd and Pb. Results showed efficient removal of Cd and Zn from the contaminated sites by the willow plants. In order to discuss the combined and long-term effect of multiple heavy metals on S. miyabeana grown in soil, variable-pressure scanning electron microscopy fitted with energy dispersive X-ray analysis (VPSEM-EDX) and SR-μ-XRF was used to characterize the serration of leaves. The combination of 2D elemental images revealed metal accumulation in the tip cells in serrations without any exudation of heavy metals from the hydathodes. © 2011 The Royal Society of Chemistry.

DOI： 10.1039/c1mt00102g

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Metabolic Engineering  

Prenylated polyphenols are secondary metabolites beneficial for human health because of their various biological activities. Metabolic engineering was performed using Streptomyces and Sophora flavescens prenyltransferase genes to produce prenylated polyphenols in transgenic legume plants. Three Streptomyces genes, NphB, SCO7190, and NovQ, whose gene products have broad substrate specificity, were overexpressed in a model legume, Lotus japonicus, in the cytosol, plastids or mitochondria with modification to induce the protein localization. Two plant genes, N8DT and G6DT, from Sophora flavescens whose gene products show narrow substrate specificity were also overexpressed in Lotus japonicus. Prenylated polyphenols were undetectable in these plants; however, supplementation of a flavonoid substrate resulted in the production of prenylated polyphenols such as 7- O-geranylgenistein, 6-dimethylallylnaringenin, 6-dimethylallylgenistein, 8-dimethylallynaringenin, and 6-dimethylallylgenistein in transgenic plants. Although transformants with the native NovQ did not produce prenylated polyphenols, modification of its codon usage led to the production of 6-dimethylallylnaringenin and 6-dimethylallylgenistein in transformants following naringenin supplementation. Prenylated polyphenols were not produced in mitochondrial-targeted transformants even under substrate feeding. SCO7190 was also expressed in soybean, and dimethylallylapigenin and dimethylallyldaidzein were produced by supplementing naringenin. This study demonstrated the potential for the production of novel prenylated polyphenols in transgenic plants. In particular, the enzymatic properties of prenyltransferases seemed to be altered in transgenic plants in a host species-dependent manner. © 2011 Elsevier Inc.

DOI： 10.1016/j.ymben.2011.07.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Geochimica et Cosmochimica Acta  

In tobacco plants, elimination of Zn and Cd via the production of Ca-containing grains at the top of leaf hairs, called trichomes, is a potent detoxification mechanism. This study examines how Cd is incorporated in these biominerals, and how calcium growth supplement modifies their nature. Scanning electron microscopy coupled with energy dispersive X-ray microanalysis (SEM-EDX), microfocused X-ray diffraction (μ-XRD), and microfocused X-ray absorption near edge structure (μ-XANES) spectroscopy were used to image the morphology of the grains, identify the crystallized mineral phases, and speciate Cd, respectively. The mineralogy of the grains and chemical form of Cd varied with the amount of Ca. When tobacco plants were grown in a nutrient solution containing 25 μM Cd and low Ca supplement (Ca/Cd = 11. mol ratio), most of the grains were oblong-shaped and low-Cd-substituted calcite. When exposed to the same amount of Cd and high Ca supplement (Ca/Cd = 131. mol ratio), grains were more abundant and diverse in compositions, and in total more Cd was eliminated. Most grains in the high Ca/Cd experiment were round-shaped and composed predominantly of Cd-substituted vaterite, a usually metastable calcium carbonate polymorph, and subordinate calcite. Calcium oxalate and a Ca amorphous phase were detected occasionally in the two treatments, but were devoid of Cd. The biomineralization of cadmium and implications of results for Cd exposure of smokers and phytoremediation are discussed. © 2010 Elsevier Ltd.

DOI： 10.1016/j.gca.2010.07.011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Plant and Cell Physiology  

Nicotiana tabacum (tobacco) plants have short and long glandular trichomes. There is evidence that tobacco trichomes play several roles in the defense against biotic and abiotic stresses. cDNA libraries were constructed from control and cadmium (Cd)-treated leaf trichomes. Almost 2,000 expressed sequence tag (EST) cDNA clones were sequenced to analyze gene expression in control and Cd-treated leaf trichomes. Genes for stress response as well as for primary metabolism scored highly, indicating that the trichome is a biologically active and stress-responsive tissue. Reverse transcriptionPCR (RTPCR) analysis demonstrated that antipathogenic T-phylloplanin-like proteins, glutathione peroxidase and several classes of pathogenesis-related (PR) proteins were expressed specifically or dominantly in trichomes. Cysteine-rich PR proteins, such as non-specific lipid transfer proteins (nsLTPs) and metallocarboxypeptidase inhibitors, are candidates for the sequestration of metals. The expression of osmotin and thaumatin-like proteins was induced by Cd treatment in both leaves and trichomes. Confocal laser scanning microscopy (CLSM) showed that glutathione levels in tip cells of both long and short trichomes were higher than those in other types of leaf cells, indicating the presence of an active sulfur-dependent protective system in trichomes. Our results revealed that the trichome-specific transcriptome approach is a powerful tool to investigate the defensive functions of trichomes against both abiotic and biotic stress. Trichomes are shown to be an enriched source of useful genes for molecular breeding towards stress-tolerant plants. © 2010 The Author.

DOI： 10.1093/pcp/pcq118

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Plant and Cell Physiology  

Trees that accumulate metals are important plants for restoring contaminated soil because of their high biomass. We examined the cadmium (Cd) tolerance and growth rate of six willow (Salix) species common in Japan. To characterize in detail the localization of Cd and its ligands, synchrotron radiation-based micro X-ray fluorescence analysis was used. This revealed the accumulation of cadmium at the tips of the serrations in leaves, and the phellogen and/or the phelloderm under the stem surface. μ-X-ray absorption near edge structure spectra of Cd in all the accumulation sites were similar to that of the Cd ion coordinated by O ligands in S. gilgiana. © 2010 The Author.

DOI： 10.1093/pcp/pcq039

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：In Vitro Cellular and Developmental Biology - Plant  

Podophyllum peltatum is an important medicinal plant that produces podophyllotoxin (PTOX) with anti-cancer properties. We established the embryogenic cell and adventitious root culture systems in P. peltatum and analyzed PTOX production. For the growth of embryogenic cell clumps in shake flask culture, the most efficient concentration of 2,4-dichloroacetic acid (2,4-D) was 6.78 μM, and the growth of embryogenic cell clumps was 15.9-fold increased in Murashige and Skoog MS liquid medium with 6.78 μM 2,4-D after 3 wk of culture. To induce adventitious roots, half-strength MS medium showed the best results for adventitious root induction compared to full strength MS medium and MS medium lacking NH4NO3. Optimal indole-3-butyric acid concentration for adventitious root formation was 14.78 μM. In liquid medium, the frequency of adventitious root formation from root segments was 87.7% and the number of laterally formed adventitious roots was 22.3 per segment. PTOX production in embryogenic cells and adventitious roots was confirmed by liquid chromatography and electrospray ionization-tandem mass spectrometry analysis. High-performance liquid chromatography analysis revealed that adventitious roots contained higher PTOX than embryogenic cell clumps. Elicitor treatment (20 μM methyl jasmonate) strongly enhanced the production of PTOX in both embryogenic cell clumps and adventitious roots. This observation suggests that both embryogenic cell and adventitious root culture can be adopted to produce PTOX. © 2008 The Society for In Vitro Biology.

DOI： 10.1007/s11627-008-9134-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Plant Biotechnology  

Tobacco (Nicotiana tabacum) was recently shown to detoxify heavy metals by exudation of metals as a metal-substituted calcite (calcium carbonate) through leaf trichomes. In this paper, we describe the applications of the variable-pressure scanning electron microscopy (VP-SEM) system to investigate tobacco trichomes exudates after heavy metal treatment. An energy-dispersive X-ray analysis (EDX) system fitted to VP-SEM revealed that the exudates contain amounts of heavy metals. Overexpression of cysteine synthase confers cadmium (Cd) tolerance to tobacco, and the endogenous concentration of Cd was 20% less in transgenic plants than in wild-type plants. We evaluated the numbers of trichomes on the leaf surfaces of wild-type and transgenic plants using VP-SEM. The numbers of both long and short trichomes in the transgenic plants were 25% higher than in that of wild-type plants, indicating the active excretion of Cd from trichomes in transgenic plants. The VPSEM-EDX system is a powerful tool to investigate plant epidermal structures and functions. Copyright © 2008 The Japanese Society for Plant Cell and Molecular Biology.

DOI： 10.5511/plantbiotechnology.25.407

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Canadian Journal of Chemistry  

Tobacco (Nicotiana tabacum L. cv. Xanthi) plants exposed to toxic levels of zinc and cadmium excrete metals through their leaf trichomes (epidermal hairs) as Zn,Ca- and Cd,Ca-containing grains. Little is known about the nature and formation mechanism of these precipitates. The chemical, crystalline, and noncrystalline compositions of individual grains produced by tobacco were studied by scanning electron microscopy coupled with energy dispersive X-ray analysis (SEM-EDX), micro-X-ray diffraction (μXRD), and calcium K-edge micro X-ray absorption near edge structure (pXANES) spectroscopy. Zinc is predominantly incorporated in calcite and cadmium in calcite and vaterite. Aragonite, which occurs occasionally, does not seem to contain trace metals. In addition to being precipitated in its three possible polymorphic forms, calcite, aragonite, and vaterite, calcium is also speciated as amorphous CaCO 3 and possibly organic Ca in some grains. Most often, a particular grain consists of two or more crystalline and noncrystalline phases. The observed variability of intra- and inter-grain elemental and phase composition suggests that this biomineralization process is not constrained by biological factors but instead results from thermodynamically and kinetically controlled reactions. This study illustrates the potential of laterally resolved X-ray synchrotron radiation techniques (μXRD and μXANES) to study biomineralization and metal immobilization processes in plants. © 2007 NRC Canada.

DOI： 10.1139/V07-076

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Plant Physiology  

Tobacco (Nicotiana tabacum L. cv Xanthi) plants were exposed to toxic levels of zinc (Zn). Zn exposure resulted in toxicity signs in plants, and these damages were partly reduced by a calcium (Ca) supplement. Confocal imaging of intracellular Zn using Zinquin showed that Zn was preferentially accumulated in trichomes. Exposure to Zn and Zn + Ca increased the trichome density and induced the production of Ca/Zn mineral grains on the head cells of trichomes. These grains were aggregates of submicrometer-sized crystals and poorly crystalline material and contained Ca as major element, along with subordinate amounts of Zn, manganese, potassium, chlorine, phosphorus, silicon, and magnesium. Micro x-ray diffraction revealed that the large majority of the grains were composed essentially of metal-substituted calcite (CaCO3). CaCO3 polymorphs (aragonite and vaterite) and CaC2O4 (Ca oxalate) mono- and dihydrate also were identified, either as an admixture to calcite or in separate grains. Some grains did not diffract, although they contained Ca, suggesting the presence of amorphous form of Ca. The presence of Zn-substituted calcite was confirmed by Zn K-edge micro-extended x-ray absorption fine structure spectroscopy. Zn bound to organic compounds and Zn-containing silica and phosphate were also identified by this technique. The proportion of Zn-substituted calcite relative to the other species increased with Ca exposure. The production of Zn-containing biogenic calcite and other Zn compounds through the trichomes is a novel mechanism involved in Zn detoxification. This study illustrates the potential of laterally resolved x-ray synchrotron radiation techniques to study biomineralization and metal homeostasis processes in plants. © 2006 American Society of Plant Biologists.

DOI： 10.1104/pp.106.082743

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Plant Biology  

Growth was severely inhibited when tobacco plants were exposed to toxic levels of cadmium (0.2 mM). However, when this treatment was combined with a high concentration of calcium (30 mM), the Cd-induced damage was strongly alleviated. Under these enhanced conditions, i.e., 30 mM Ca and 0.2 mM Cd, Ca crystals not only were heavily deposited in the leaves but were also actively excreted from the trichomes. The X-ray spectrum from our Energy Dispersive analysis revealed that both intra- and extracellular Ca crystals contained detectable amounts of Cd. Moreover, intracellular Ca deposition in the leaves was stimulated only by a high Ca concentration (30 mM); moderate levels of Ca (3 mM) or a toxic amount of Cd (0.2 mM) alone resulted in crystal deposition that was undetectable under a light microscope. In contrast, extracellular crystal formation on the trichomes was stimulated by toxic Cd treatment but not by high Ca concentrations alone. Finally, Inductively Coupled Plasma Spectroscopy revealed that a high level of Ca (30 mM) suppressed Cd accumulation while also increasing the endogeneous Ca concentration in the leaves. These observations imply that the amelioration of Ca against toxic Cd in tobacco plants is a result of not only the inhibition of Cd uptake, but also the extra- and intracellular sequestration of cadmium via Ca crystallization.

DOI： 10.1007/BF03030570

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Phytochemistry  

Squalene synthase (SS) catalyzes the first committed step in sterol and triterpenoid biosynthesis. Transgenic Eleutherococcus senticosus Rupr. and Maxim. plants were generated by introducing an SS-encoding gene derived from Panax ginseng (PgSS1) together with genes expressing hygromycin phosphotransferase and green fluorescent protein (GFP) through Agrobacterium-mediated transformation. Early globular embryo clusters developing from the embryogenic callus were used for Agrobacterium-mediated transformation. Transformants were selected on Murashige Skoog medium containing 25 mg/L hygromycin. Hygromycin-resistant somatic embryos developed into plants after the cotyledonary embryos were treated with 14.4 μM gibberellic acid. Transformation was confirmed by polymerase chain reaction, Southern, and GFP analyses. The SS enzyme activity of the transgenic plants was up to 3-fold higher than that of wild-type plants. In addition, GC-MS and HPLC analysis revealed that phytosterols (β-sitosterol and stigmasterol) as well as triterpene saponins (ciwujianosides B (1), C1 (2), C2 (3), C3 (4), C4 (5), D1 (6) and D2 (7)) levels in transgenic E. senticosus were increased by 2- to 2.5-fold. These results suggest that the metabolic engineering of E. senticosus to enhance production of phytosterols and triterpenoids by introducing the PgSS1 gene was successfully achieved by Agrobacterium-mediated genetic transformation. © 2005 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.phytochem.2005.02.016

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Phytochemistry  

Phytochelatins are essential for metal detoxification in plants, fungi and nematodes. Phytochelatin synthase-related proteins apparently exist also in organisms not known to produce phytochelatins. We found for a cyanobacterial protein that it lacks phytochelatin synthase activity. Instead, it catalyzes the glutathione to γ-glutamylcysteine conversion. Our data suggest the evolutionary origin of phytochelatin synthesis. Phytochelatins are glutathione-derived, non-translationally synthesized peptides essential for cadmium and arsenic detoxification in plant, fungal and nematode model systems. Recent sequencing programs have revealed the existence of phytochelatin synthase-related genes in a wide range of organisms that have not been reported yet to produce phytochelatins. Among those are several cyanobacteria. We have studied one of the encoded proteins (alr0975 from Nostoc sp. strain PCC 7120) and demonstrate here that it does not possess phytochelatin synthase activity. Instead, this protein catalyzes the conversion of glutathione to γ-glutamylcysteine. The thiol spectrum of yeast cells expressing alr0975 shows the disappearance of glutathione and the formation of a compound that by LC-MSMS analysis was unequivocally identified as γ-glutamylcysteine. Purified recombinant protein catalyzes the respective reaction. Unlike phytochelatin synthesis, the conversion of glutathione to γ- glutamylcysteine is not dependent on activation by metal cations. No evidence was found for the accumulation of phytochelatins in cyanobacteria even after prolonged exposure to toxic Cd 2+ concentrations. Expression of alr0975 was detected in Nostoc sp. cells with an antiserum raised against the protein. No indication for a responsiveness of expression to toxic metal exposure was found. Taken together, these data provide further evidence for possible additional functions of phytochelatin synthase-related proteins in glutathione metabolism and provide a lead as to the evolutionary history of phytochelatin synthesis. © 2004 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.phytochem.2004.09.017

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Plant Biology  

When tobacco (Nicotiana tabacum) plants are exposed to toxic level of cadmium (0.2 mM Cd), their trichomes actively excrete crystals (Choi et al., 2001). In this study, we investigated the distribution of Cd and NaCl on trichomes and leaf surfaces. Energy dispersive x-ray (EDX) analysis revealed that, under toxic Cd stress, crystals exudated from the trichomes contained high amounts of Ca, Mg, and Cd, as well as low levels of P, S, and Mn. Electron spectroscopic imaging (ESI) from trichomes and attached crystals showed that these crystals emitted denser radiation energy for Ca and Cd than did the head cells of the trichomes. However, no Cd was detected on the trichome surface itself or within the leaf epidermis. In contrast, treatment with salt (NaCl) did not stimulate crystal formation; instead, it induced the abnormal expansion of trichome cells. Although Na was not accumulated within the crystals, a considerable amount of both Na and Cl was sequestered within the stalk cells of the long trichomes. Therefore, we believe that tobacco trichomes play an important role in Cd crystal exudation through crystallization, but that, under NaCl stress, the long trichomes sequester those elements within their stalks.

DOI： 10.1007/BF03030635

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Plant Journal  

The hyperaccumulation of zinc (Zn) and cadmium (Cd) is a constitutive property of the metallophyte Arabidopsis halleri. We therefore used Arabidopsis GeneChips to identify genes more active in roots of A. halleri as compared to A. thaliana under control conditions. The two genes showing highest expression in A. halleri roots relative to A. thaliana roots out of more than 8000 genes present on the chip encode a nicotianamine (NA) synthase and a putative Zn 2+ uptake system. The significantly higher activity of these and other genes involved in metal homeostasis under various growth conditions was confirmed by Northern and RT-PCR analyses. A. halleri roots also show higher NA synthase protein levels. Furthermore, we developed a capillary liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (CapLC-ESI-QTOF-MS)-based NA analysis procedure and consistently found higher NA levels in roots of A. halleri. Expression of a NA synthase in Zn2+-hypersensitive Schizosaccharomyces pombe cells demonstrated that formation of NA can confer Zn2+ tolerance. Taken together, these observations implicate NA in plant Zn homeostasis and NA synthase in the hyperaccumulation of Zn by A. halleri. Furthermore, the results show that comparative microarray analysis of closely related species can be a valuable tool for the elucidation of phenotypic differences between such species.

DOI： 10.1046/j.1365-313X.2003.01960.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Plant Physiology  

One of the defense systems against toxic heavy metals in plants is to employ cysteine-rich chelating compounds, phytochelatins, which are polymeric forms of glutathione. In the present study cadmium stressed Arabidopsis was employed in order to investigate the response of genes involved in glutathione biosynthesis via cysteine and production of thiol compounds. The results showed a 13-fold increase in transcripts for ATP sulfurylase, 6 to 10-fold for APS reductase and 2-fold for sulfite reductase, all of which are involved in cysteine synthesis. In addition, a 3-fold increase in total thiols was noted, mainly of glutathione, phytochelatins and cysteine. It is suggested that, to cope with toxic heavy metals, plants activate the sulfur assimilation pathway by increasing transcription of related genes to provide an enhanced supply of glutathione for phytochelatin biosynthesis.

DOI： 10.1078/0176-1617-00733

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Planta  

In tobacco (Nicotiana tabacum L.), long and short trichomes can be distinguished morphologically. The established function of long trichomes is to exude a sticky gum containing diterpenes, whereas that of short trichomes is not known. When tobacco seedlings were exposed to toxic levels of cadmium (Cd), growth was retarded, but trichome number was increased up to 2-fold in comparison with untreated samples. Observation by variable-pressure scanning electron microscopy (VP-SEM) indicated that large crystals of 150 μm in size were formed on head cells of both short and long trichomes. An energy-dispersive X-ray analysis system fitted with VP-SEM revealed the crystals to contain amounts of Cd and calcium (Ca) at much higher concentrations than in the head cells themselves. Transmission electron microscopy demonstrated crystal formation in amorphous osmiophilic deposits in vacuoles. When seedlings were treated with Cd in the presence of Ca, tolerance was increased in proportion to the increase in Ca concentration. These results indicate that tobacco plants actively exclude toxic Cd by forming and excreting Cd/Ca-containing crystals through the head cells of trichomes.

DOI： 10.1007/s004250000487

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Language：Japanese   Publishing type：Thesis (other)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Plant Physiology  

Plants tolerate heavy metals through sequestration with cysteine-rich peptides, phytochelatins. In this reaction, the rate limiting step is considered to be the supply of cysteine, which is synthesized by cysteine synthase (CS, EC 4.2.99.8) from hydrogen sulfide and O-acetylserine. In this study, we transformed tobacco (Nicotiana tabacum) plants with RCS1, a cytosolic cysteine synthase gene of rice (Oryza sativa), and examined their sensitivity to cadmium. The transgenic plants had up to 3-fold higher activity of cysteine synthase than wild-type plants. Upon exposure to cadmium, they exhibited obvious tolerance with much greater growth than wild-type plants. The level of phytochelatins in shoots was higher in transgenic than in wild-type plants after cadmium treatment, suggesting that cadmium was actively trapped by phytochelatins. However, the cadmium concentration per g fresh weight of whole transgenic plants was 20 % lower than that of wild-type plants, suggesting cadmium to be either actively excreted or diluted by fast growth. Genetic analysis of progenies clearly showed segregation of cadmium tolerance, indicating that the trait resulted from the introduced gene. These results suggest that introduction of a cysteine synthase gene into tobacco plants resulted not only in high level production of sulfur-containing compounds that detoxify cadmium, but also in active elimination of cadmium toxicity from plant bodies.

DOI： 10.1078/0176-1617-00314

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Plant Physiology  

Effects of mechanical wounding on levels of transcripts encoding enzymes involved in the sulfur assimilation pathway were examined. RNA blot hybridization showed the sulfate transporter, ATP sulfurylase, adenosine phosphosulfate (APS) kinase, APS reductase and cysteine synthase, all but the first being chloroplastic isozymes differentially responding to wounding, and also to jasmonate, a powerful inducer of wound responsive gene expression. When plants were exposed to jasmonate, transcripts for APS kinase were increased within 1 h after treatment, while those for APS reductase were increased after 3 h. We also found the glucosinolate content to be 2-fold elevated at the latter time point. APS is directed to the secondary metabolic pathway by APS kinase, so that glucosinolates are synthesized, while it is allotted through the action of APS reductase to the primary metabolic pathway by which cysteine is synthesized. Consequently, our results suggest that wounded plants primarily deliver available sulfur to synthesize defense-related substances by activating genes involved in the secondary metabolic pathway.

DOI： 10.1016/S0176-1617(00)80317-X

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Bioscience, Biotechnology and Biochemistry  

Expression of nine genes encoding enzymes involved in the sulfur assimilation pathway was examined by RNA blot hybridization. Significantly increased levels of transcripts encoding ATP sulfurylase and APS reductase were apparent under sulfur deprivation. However, in the absence of nitrogen, their responsiveness to sulfur deprivation was markedly reduced. Results suggest that the sulfur assimilation pathway is regulated at the transcriptional level by both nitrogen and sulfur sources. © 1999, Taylor & Francis Group, LLC. All rights reserved.

DOI： 10.1271/bbb.63.762

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Yakugaku Zasshi  

During the course of our studies on the chemical constituents of botanically identified Chinese licorice roots of various origins, we have identified 15 kinds of saponins and 49 kinds of flavonoids (including flavonoid glycosides). With these chemical constituents at hand, we have carried out the chemical evaluation studies of licorice roots available in Japan by means of high performance liquid chromatography (HPLC). In the HPLC analysis using an ODS reversed-phase column, a combination of 1% aqueous acetic acid and acetonitrile was used as the mobile phase in a gradient solvent system, and the eluate was monitored with UV absorption at 254 nm (for saponins with a diene or a conjugated carbonyl moiety, liquiritin, and liquiritin apioside) or at 350 nm (for other flavonoid constituents). By our analytical method described here, quantitative analyses of saponin and flavonoid constituents were effected simultaneously. We have found an interesting piece of information concerning the origin of parent plants and their characteristic constituents such as saponins and flavonoids.

DOI： 10.1248/yakushi1947.118.11_519

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Chemical and Pharmaceutical Bulletin  

Potent inhibitors of ethanol absorption, elatosides A and B, were isolated from the bark of Aralia elata SEEM. through bioassay-guided separation together with elatosides C and D and four known oleanolic acid glucuronide-saponins, spinasaponin A, spinasaponin A 28-O-glucoside, and stipuleanosides R1 and R2. The structures of elatosides A, B, C, and D were determined on the basis of chemical and physicochemical evidence as oleanolic acid 3-O-{[β-D-xylopyranosyl (1→2)] [β-D-galactopyranosyl (1→3)]}-β- D-glucopyranosiduronic acid, oleanolic acid 3-O-{[β-D-galactopyranosyl (1- 2)] [β-D-galactopyranosyl (1-3)]}-β-D-glucopyranosiduronic acid, and their 28-O-glucopyranosyl esters, respectively. The inhibitory effect of various oleanolic acid 3, 28-O-bisdesmosides, oleanolic acid 3-O-monodesmosides, and oleanolic acid on ethanol absorption was examined and it was found that the 3-O-glucuronide moiety and the 28-carboxyl group in oleanolic acid glucuronide-saponin were required to exert the inhibitory activity.

DOI： 10.1248/cpb.44.1915

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Chemical and Pharmaceutical Bulletin  

The hypoglycemic component, elatoside E, was isolated from the root cortex of Aralia elata SEEM. (Araliaceae) together with elatoside F and eight known oleanolic acid glycosides, elatosides A and C, oleanolic acid 3-O-[α- L-arabinofuranosyl (1→4)]-β-D-glucopyranosiduronic acid, oleanolic acid 3- O-β-D-glucopyranosiduronic acid, stipuleanosides R1 and R2, and chikusetsusaponins IV and IVa. The structures of elatosides E and F were determined on the basis of chemical and physicochemical evidence as oleanolic acid 3-O-[β-D-xylopyranosyl (1→2)][β-D-glucopyranosyl (1→3)]-α-L- arabinopyranoside and its 28-O-β-D-glucopyranosyl ester, respectively. The hypoglycemic activity of oleanolic acid and nine oleanolic acid oligoglycosides from the root cortex of Aralia elata was determined by monitoring inhibition effect on the elevation of plasma glucose level by oral sucrose tolerance test in rats, and some structure-activity relationships of oleanolic acid glycoside were obtained.

DOI： 10.1248/cpb.44.1923

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Yakugaku Zasshi  

In the course of our search for natural antioxidants, the methanol extract of the fruit hulls of mangosteen (Garcinia mangostana L.) originating in Vietnam was found to exhibit a potent radical scavenging effect. By monitoring the radical scavenging effect, two xanthones, α- and γ- mangostins, were isolated together with (-)-epicatechin, procyanidins A-2 and B-2 as active principles. The antioxidative activity of these two xanthones was measured by the ferric thiocyanate method and it was found that γ- mangostin showed more potent antioxidative activity than BHA and α- tocopherol.

DOI： 10.1248/yakushi1947.114.2_129

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Chemical and Pharmaceutical Bulletin  

New type inhibitors of ethanol absorption, camelliasaponins B1, B2, C1 and C2, were isolated from the seeds of Camellia japonica L. The structures of camelliasaponins were elucidated on the basis of chemical and physicochemical evidence. The inhibitory effect of camelliasaponins and related saponins on ethanol absorption have been examined, and it was found that the triterpene oligoglycoside structure having an acyl group was essential to exerting the activity. © 1994, The Pharmaceutical Society of Japan. All rights reserved.

DOI： 10.1248/cpb.42.742

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Yakugaku Zasshi  

In order to characterize the chemical change of the constituents during the processing of Hydrangeae Dulcis Folium, quantitative analyses of phyllodulcin, hydrangenol, and their 8-O-glucosides were developed by means of high performance liquid chromatography. As an application of this HPLC method, the distribution of those dihydroisocoumarins in different parts of Hydrangea macrophylla var. thunbergii was investigated. It was found that these dihydroisocoumarins were contained at the highest concentration in the leaves. Furthermore, the seasonal fluctuation of these compounds in the leaves, together with the height of the plant and total dry weight of the leaves, were clarified and so that, the suitable period for the harvest of Hydrangea macrophylla var. thunbergii was deduced to be from Oct. to Nov.

DOI： 10.1248/yakushi1947.114.3_176

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Chemical and Pharmaceutical Bulletin  

Five triterpene oligoglycosides named escins-Ia, Ib, IIa, IIb, and IIIa were isolated from the seeds of Aesculus hippocastanum L. and their chemical structures were determined on the basis of chemical and physicochemical evidence. Escins-Ia, Ib, IIa, and lib were found to exhibit inhibitory effect on ethanol absorption and hypoglycemic activity on oral glucose tolerance test in rats. Among them, escins-IIa and IIb showed the higher activities for both bioassays, while desacylescins-I and II had no activity. © 1994, The Pharmaceutical Society of Japan. All rights reserved.

DOI： 10.1248/cpb.42.1357

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Chemical and Pharmaceutical Bulletin  

A new inhibitor named elatosides E (which was shown to affect the elevation of plasma glucose level by oral sugar tolerance test in rats) was isolated from the root cortex of Aralia elata Seem, together with elatoside F. The structures of elatosides E and F were elucidated on the basis of chemical and physicochemical evidence. The hypoglycemic activities of oleanolic acid and nine oleanolic acid glycosides obtained from the root cortex of Aralia elata have been examined, and some structure-activity relationships have been found. © 1994, The Pharmaceutical Society of Japan. All rights reserved.

DOI： 10.1248/cpb.42.1354

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Chemical and Pharmaceutical Bulletin  

From the less polar fraction of Hydrangeae Dulcis Folium, the fermented and dried leaves of Hydrangea macrophylla Seringe var. thunbergii Makino, eight antiallergic and antimicrobial principles were isolated together with several known compounds. Among the newly isolated bioactive constituents, the chemical structures of thunberginols A, B, and F have been determined on the basis of chemical and physicochemical evidence. Thunberginols A, B, and F were found to exhibit more potent antiallergic activity than phyllodulcin, hydrangenol, disodium cromoglycate (DSCG), and tranilast. In addition, these thunberginols showed antimicrobial activity against oral bacteria. © 1994, The Pharmaceutical Society of Japan. All rights reserved.

DOI： 10.1248/cpb.42.2225

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Yakugaku Zasshi  

In order to develop new bioactive functions of Hydrangeae Dulcis Folium, the fermented and dried leaves of Hydrangea macrophylla Seringe var. thunbergii Makino, effects of the methanolic extract from the crude drug on antiulcer, antiallergic, cholagoic, and various pharmacological actions were investigated. Consequently, the methanolic extract was found to exhibit potent antiulcer, antiallergic, and cholagoic activities. By monitoring with these activities, it was found that the active constituents were contained in the lipophilic portion of the methanolic extract. Furthermore, the known lipophilic constituents such as phyllodulcin and hydrangenol were found to show little antiulcer and cholagoic activities, while it was also found that they showed antiallergic activity on Schultz-Dale reactions.

DOI： 10.1248/yakushi1947.114.6_401

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Chemical and Pharmaceutical Bulletin  

Oxidative lactonization of 2-carboxystilbene mediated by CuCl2 proceeded regiospecifically to give the five-membered lactone. By utilizing this lactonization as a key reaction, chemical transformation from dihydroisocoumarine into benzylidencphthalide was accomplished, and it was applied to structural elucidation of two new phthalide, hydramacroohyllols A and B. © 1994, The Pharmaceutical Society of Japan. All rights reserved.

DOI： 10.1248/cpb.42.721

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Chemical and Pharmaceutical Bulletin  

Two new labile monoterpenes named paeonisuffrone and paeonisuffral were isolated from Chinese Moutan Cortex, the dried root of Paeonia suffruticosa ANDREWS. The absolute stereostructures of paeonisuffrone and paeonisuffral were elucidated on the basis of chemical and physicochemical evidence which included the application of a modified Mosher’s method and lipase catalyzed debenzoylation. © 1993, The Pharmaceutical Society of Japan. All rights reserved.

DOI： 10.1248/cpb.41.630

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Chemical and Pharmaceutical Bulletin  

By monitoring the inhibitory effect on ethanol absorption in rats, new active saponins named elatosides A and B were isolated from the bark of Aralia elata seem, together with elatosides C and D. The structures of elatosides A, B, C, and D were elucidated on the basis of chemical and physicochemical evidence. The inhibitory effects of several oleanolic acid oligoglycosides on ethanol absorption have been examined and some structure-activity relationships have been found. © 1993, The Pharmaceutical Society of Japan. All rights reserved.

DOI： 10.1248/cpb.41.2069

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Chemical and Pharmaceutical Bulletin  

Two new quassinoids named picrajavanins A and B were isolated, together with known β-carboline alkaloids and picrasidine G, from the bark of Picrasma javanica bl. (Simaroubaceae), an Indonesian folk medicine, collected on Flores Island. The absolute stereostructures of picrajavanins A (6) and B (7) have been elucidated on the basis of chemical and physicochemical evidence, together with the application of the modified Mosher’s method. © 1993, The Pharmaceutical Society of Japan. All rights reserved.

DOI： 10.1248/cpb.41.2101

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Chemical and Pharmaceutical Bulletin  

Six new antiallergic and antimicrobial principles, thunberginols A, B, C, D, E, and F, were isolated from Hydrangeae Dulcis Folium, the fermented and dried leaves of Hydrangea macrophylla SERINGE var. thunbergii MAKINO. The chemical structures of thunberginols A, B, and F have been determined on the basis of chemical and physicochemical evidence. Thunberginols A, B, and F showed more potent antiallergic activity than phyllodulcin, hydrangenol, and AA-861 in the in vitro test using the Schults-Dale reaction in sensitized guinea pig bronchial muscle. Thunberginols A, B, and F also exhibited antimicrobial activity against oral bacteria. © 1992, The Pharmaceutical Society of Japan. All rights reserved.

DOI： 10.1248/cpb.40.3121

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Chemical and Pharmaceutical Bulletin  

New antiallergic and antimicrobial dihydroisocoumarins, thunberginols C, D, and E, were isolated from Hydrangeae Dulcis Folium, the fermented and dried leaves of Hydrangea macrophylla SERINGE var. thunbergii MAKINO, together with new dihydroisocoumarin glycosides, thunberginol G 3’-0-glucoside and (-)-hydrangenol 4’-0-glucoside. Their chemical structures have been determined on the basis of chemical and physicochemical evidence. Thunberginols C, D, E, G, and (-)-hydrangenol 4’-0-glucoside showed antiallergic activity in the in vitro bioassay using the Schults-Dale reaction in sensitized guinea pig bronchial muscle, and they also exhibited antimicrobial activity against oral bacteria. © 1992, The Pharmaceutical Society of Japan. All rights reserved.

DOI： 10.1248/cpb.40.3352

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