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.

Nanobubbles in vase water inhibit transpiration and prolong the vase life of cut chrysanthemum flowers.

Nanobubble (NB) water has been shown to promote the growth of several types of plants and animals, but the mechanism underlying this promoting effect remains unclear. The present study evaluated the mechanism by which NBs maintain the freshness of cut flowers by keeping cut chrysanthemum (Chrysanthemum morifolium Ramat.) flowers at the bud stage in vase water containing air NBs. The condition of petals and leaves was assessed to determine the vase life of these cut flowers. The NB treatment delayed bud opening and petal senescence of the inflorescences. Water absorption and transpiration by cut flower stems were lower in NB water than in distilled water (DW). Furthermore, when all the leaves were removed from the cut flower stems, no significant difference in vase life was observed between NB water and DW. These findings indicate that the inhibition of transpiration from leaves prolonged the vase life of NB-treated cut chrysanthemum flowers. In the early stage of the treatment, NB treatment significantly reduced transpiration without closing stomata, suggesting that the reduction in transpiration observed in the NB-treated plants might be due to the suppression of cuticular transpiration, defined as water loss through the epidermis. Surface tension, one of the important driving forces of water movement in plants, was not affected by the presence of NBs in water. To our knowledge, this is the first report to show that transpiration from leaves is inhibited by NB treatment.

Phylogenetic, population structure, and population demographic analyses reveal that Vicia sepium in Japan is native and not introduced.

Vicia sepium (bush vetch) is a perennial legume widely distributed throughout the Eurasian continent. However, its distribution in Japan is limited to Mt. Ibuki and small parts of central and southern Hokkaido. Therefore, each Japanese V. sepium lineage has been considered to have been introduced separately from Europe. Here, we examined whether the species was introduced or not on the basis of cpDNA sequences and genome-wide SNPs from Japanese and overseas samples. Both the cpDNA haplotype network and the nuclear DNA phylogenetic tree showed that Japanese V. sepium is monophyletic. Furthermore, although the nuclear DNA phylogenetic tree also showed that each lineage is clearly monophyletic, genetic admixture of the genetic cluster dominated in the Hokkaido lineage was also detected in the Mt. Ibuki lineage. Population divergence analysis showed that the two lineages diverged during the last glacial period. The Mt. Ibuki lineage showed a sudden population decline 300-400 years ago, indicating that some anthropogenic activity might be involved, while the Hokkaido lineage showed a gradual population decline from 5000 years ago. Consequently, these two lineages show low current genetic diversity compared with overseas lineages. These results show that the Japanese V. sepium is not introduced but is native.

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.

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.

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.

Structural element responsible for the Fe(III)-phytosiderophore specific transport by HvYS1 transporter in barley.

Hordeum vulgare L. yellow stripe 1 (HvYS1) is a selective transporter for Fe(III)-phytosiderophores, involved in primary iron acquisition from soils in barley roots. In contrast, Zea mays yellow stripe 1 (ZmYS1) in maize possesses broad substrate specificity, despite a high homology with HvYS1. Here we revealed, by assessing the transport activity of a series of HvYS1-ZmYS1 chimeras, that the outer membrane loop between the sixth and seventh transmembrane regions is essential for substrate specificity. Circular dichroism spectra indicated that a synthetic peptide corresponding to the loop of HvYS1 forms an alpha-helix in solution, whereas that of ZmYS1 is flexible. We propose that the structural difference at this particular loop determines the substrate specificity of the HvYS1 transporter.

Overexpression of squalene synthase in Eleutherococcus senticosus increases phytosterol and triterpene accumulation.

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 microM 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 (beta-sitosterol and stigmasterol) as well as triterpene saponins (ciwujianosides B (1), C(1) (2), C(2) (3), C(3) (4), C(4) (5), D(1) (6) and D(2) (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.

A cyanobacterial protein with similarity to phytochelatin synthases catalyzes the conversion of glutathione to gamma-glutamylcysteine and lacks phytochelatin synthase activity.

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 gamma-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 gamma-glutamylcysteine. Purified recombinant protein catalyzes the respective reaction. Unlike phytochelatin synthesis, the conversion of glutathione to gamma-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 Cd2+ 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.

Assessment of willow (Salix sp.) as a woody heavy metal accumulator: field survey and in vivo X-ray analyses.

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.

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.

Comparative microarray analysis of Arabidopsis thaliana and Arabidopsis halleri roots identifies nicotianamine synthase, a ZIP transporter and other genes as potential metal hyperaccumulation factors.

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 Zn2+ 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.