Emiko Harada

Trichomes of Tobacco Excrete Zinc as Zinc-Substituted Calcium Carbonate and Other Zinc-Containing Compounds

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.

Expression Profiling of Tobacco Leaf Trichomes Identifies Genes for Biotic and Abiotic Stresses

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 transcription-PCR (RT-PCR) 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.

Metabolic engineering for the production of prenylated polyphenols in transgenic legume plants using bacterial and plant prenyltransferases.

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.

Characterization of Cadmium Accumulation in Willow as a Woody Metal Accumulator Using Synchrotron Radiation-Based X-Ray Microanalyses

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. micro-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.

Metal accumulation by Arabidopsis halleri subsp. gemmifera at a limestone mining site

Arabidopsis halleri subsp. gemmifera, a close wild relative of A. thaliana in eastern Asia, is an important model species of Brassicaceae used to study cadmium (Cd) and zinc (Zn) tolerance and hyperaccumulation in plants. To investigate the effects of soil factors on metal accumulation in this plant, we collected plants and rhizosphere soil samples from a limestone mining site on Mount Ibuki, and compared them with those collected from non-calcareous soil in Japan. Irrespective of the sampling site, all the plants efficiently accumulated Cd in shoot tissues. The plants growing on non-calcareous soil also accumulated Zn in shoot tissues, but shoot Zn concentration in plants growing on calcareous soils was below the level required for hyperaccumulators. The pH of calcareous soil samples was between 7.68 and 8.21. Total Zn contents were similar in calcareous and non-calcareous soils, but the amounts of Zn extractable by 0.1 M HCl were lower in calcareous than in non-calcareous soil. These results indicate that the properties of calcareous soil affect metal accumulation in plants.

Radiocesium accumulation in Egeria densa, a submerged plant – possible mechanism of cesium absorption

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.

Mn accumulation in a submerged plant Egeria densa (Hydrocharitaceae) is mediated by epiphytic bacteria

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.

Synchrotron Radiation X-Ray Analysis of Metal-Accumulating Plants

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.