Ozone-Sensitive Arabidopsis Mutants with Deficiencies in Photorespiratory Enzymes.

An ozone-sensitive mutant was isolated from T-DNA-tagged lines of Arabidopsis thaliana. The T-DNA was inserted at a locus on chromosome 3, where two genes encoding glycolate oxidases, GOX1 and GOX2, peroxisomal enzymes involved in photorespiration, reside contiguously. The amounts of the mutant's foliar transcripts for these genes were reduced, and glycolate oxidase activity was approximately 60% of that of the wild-type plants. No difference in growth and appearance was observed between the mutant and the wild-type plants under normal conditions with ambient air under a light intensity of 100 µmol photons m-2 s-1. However, signs of severe damage, such as chlorosis and ion leakage from the tissue, rapidly appeared in mutant leaves in response to ozone treatment at a concentration of 0.2 µl l-1 under a higher light intensity of 350 µmol photons m-2 s-1 that caused no such symptoms in the wild-type plant. The mutant also exhibited sensitivity to sulfur dioxide and long-term high-intensity light. Arabidopsis mutants with deficiencies in other photorespiratory enzymes such as glutamate:glyoxylate aminotransferase and hydroxypyruvate reductase also exhibited ozone sensitivities. Therefore, photorespiration appears to be involved in protection against photooxidative stress caused by ozone and other abiotic factors under high-intensity light.

Study on the relationship between the dispersal of wild boar (Sus scrofa) and the associated variability of Cesium-137 concentrations in its muscle Post-Fukushima Daiichi Nuclear Power Plant accident.

After the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident in 2011, the wild boar (Sus scrofa) population within the Fukushima Evacuation Zone (FEZ) increased substantially in size and distribution. This growing population and their potential dispersal from the FEZ, where they are exposed to high levels of radionuclides, into the surrounding landscape underscores the need to better understand boar movement patterns in order to establish policies for managing shipping restrictions for boar meat and develop management strategies. In this study, we quantified the genetic population structure of boar in and around Fukushima prefecture using sequence data of the mitochondrial DNA control region and MIG-seq analysis using 348 boar samples to clarify boar dispersal patterns. Among boar samples, seven Asian haplotypes and one European haplotype were detected. The European haplotype originated from hybridization between domestic pigs and native boar in the evacuation zone after the accident and was detected in 15 samples across a broad geographic area. Our MIG-seq analysis revealed genetic structure of boar was significantly different between boar inhabiting the eastern (including FEZ. i.e., East clade) and western (i.e., West clade) regions in Fukushima prefecture. In addition, we investigated the relationships between boar dispersal and Cesium (Cs)-137 activity concentrations in boar muscle using MIG-seq genetic data in Nihonmatsu city, located in the central-northern region of Fukushima. High Cs-137 activity concentrations, exceeding 1000 Bq/kg, in boar muscle had a significantly high probability of belonging to the East clade within localized regions. Thus, our results provide evidence of the spatial scale of dispersal of individuals or offspring of boar from the FEZ. Results of this research also indicate that dispersal of individuals between areas with different Cs-137 contamination levels is one of the biggest factors contributing to variation in Cs-137 activity concentration in boar muscle within localized regions.

Exploring simple ways to avoid collecting highly 137Cs-contaminated Aralia elata buds for the revival of local wild vegetable cultures.

Collection and cooking of wild vegetables have provided seasonal enjoyments for Japanese local people as provisioning and cultural ecosystem services. However, the Fukushima Daiichi Nuclear Power Plant accident in March 2011 caused extensive radiocesium contamination of wild vegetables. Restrictions on commercial shipments of wild vegetables have been in place for the last 10 years. Some species, including buds of Aralia elata, are currently showing radiocesium concentrations both above and below the Japanese reference level for food (100 Bq/kg), implying that there are factors decreasing and increasing the 137Cs concentration. Here, we evaluated easy-to-measure environmental variables (dose rate at the soil surface, organic soil layer thickness, slope steepness, and presence/absence of decontamination practices) and the 137Cs concentrations of 40 A. elata buds at 38 locations in Fukushima Prefecture to provide helpful information on avoiding collecting highly contaminated buds. The 137Cs concentrations in A. elata buds ranged from 1 to 6,280 Bq/kg fresh weight and increased significantly with increases in the dose rate at the soil surface (0.10-6.50 µSv/h). Meanwhile, the 137Cs concentration in A. elata buds were not reduced by decontamination practices. These findings suggest that measuring the latest dose rate at the soil surface at the base of A. elata plants is a helpful way to avoid collecting buds with higher 137Cs concentrations and aid in the management of species in polluted regions.

Explaining the variation in 137Cs aggregated transfer factor for wild edible plants as a case study on Koshiabura (Eleutherococcus sciadophylloides) buds.

The aggregated transfer factor (Tag) is commonly used to represent the actual transfer of radiocesium from soil to wild edible plants, but the values have shown substantial variation since the Fukushima nuclear accident. To elucidate the factors causing this variation, we investigated the effects of spatial scale and vertical 137Cs distribution in the soil on the variation of Tag-137Cs values for one of the most severely contaminated wild edible plants, Eleutherococcus sciadophylloides Franch. et Sav. (Koshiabura). The variation in Tag-137Cs values was not reduced by direct measurement of 137Cs deposition in soil samples from the Koshiabura habitat, as a substitute for using spatially averaged airborne survey data at the administrative district scale. The 137Cs activity concentration in Koshiabura buds showed a significant positive correlation with the 137Cs inventories only in the organic horizon of soil from the Koshiabura habitat. The ratio of 137Cs inventories in the organic horizon to the total 137Cs deposition in soil exhibited substantial variation, especially in broad-leaved deciduous forests that Koshiabura primarily inhabits. This variation may be the cause of the wide range of Tag-137Cs values observed in Koshiabura buds when calculated from the total 137Cs deposition in soil.

The integral membrane protein SEN1 is required for symbiotic nitrogen fixation in Lotus japonicus nodules.

Legume plants establish a symbiotic association with bacteria called rhizobia, resulting in the formation of nitrogen-fixing root nodules. A Lotus japonicus symbiotic mutant, sen1, forms nodules that are infected by rhizobia but that do not fix nitrogen. Here, we report molecular identification of the causal gene, SEN1, by map-based cloning. The SEN1 gene encodes an integral membrane protein homologous to Glycine max nodulin-21, and also to CCC1, a vacuolar iron/manganese transporter of Saccharomyces cerevisiae, and VIT1, a vacuolar iron transporter of Arabidopsis thaliana. Expression of the SEN1 gene was detected exclusively in nodule-infected cells and increased during nodule development. Nif gene expression as well as the presence of nitrogenase proteins was detected in rhizobia from sen1 nodules, although the levels of expression were low compared with those from wild-type nodules. Microscopic observations revealed that symbiosome and/or bacteroid differentiation are impaired in the sen1 nodules even at a very early stage of nodule development. Phylogenetic analysis indicated that SEN1 belongs to a protein clade specific to legumes. These results indicate that SEN1 is essential for nitrogen fixation activity and symbiosome/bacteroid differentiation in legume nodules.

High-throughput capture of nucleotide sequence polymorphisms in three Brassica species (Brassicaceae) using DNA microarrays.

PREMISE OF THE STUDY: To capture molecular markers that are applicable to environmental risk assessment of genetically modified oilseed rape, and to streamline their development, we screened variations in nucleotide sequences of three Brassica species by DNA microarray analysis. METHODS AND RESULTS: Using the Affymetrix GeneChip Arabidopsis ATH1 Genome Array, we monitored gene expression at 22810 loci among the Brassica species and picked out 192 putative polymorphic loci. We sequenced 25 of these and successfully aligned them among all three species. All 25 loci possessed some interspecific and at times intraspecific nucleotide variation. CONCLUSIONS: DNA microarray analysis effectively detected a large number of nucleotide sequence variations among closely related Brassica species. The polymorphic regions will allow the subsequent development of functional gene markers.

Monitoring of radioactive cesium in wild boars captured inside the difficult-to-return zone in Fukushima Prefecture over a 5-year period.

Following the Fukushima Daiichi Nuclear Power Plant accident in 2011, tissue samples from wild boar (Sus scrofa) outside the evacuation zone (difficult-to-return zone, DRZ) tended to show high activity concentrations of cesium-137 (137Cs). Understanding the 137Cs dynamics of wild boar populations inside the DRZ is necessary because they affect 137Cs dynamics and wild boar management in areas outside the DRZ. Since few detailed, long-term studies have been conducted inside the DRZ, we measured 137Cs activity concentrations in 221 wild boar muscle samples obtained from wild boar caught inside the DRZ and surrounding areas over a 5-year period. Our results showed that the 137Cs activity concentration in wild boar from inside the DRZ were higher than those in wild boar outside this zone. No significant difference was observed between muscle and soil 137Cs levels, but significant correlations were observed between muscle 137Cs activity concentrations and body length and body weight in the low-activity-concentration season, but not between all seasons and the high-activity-concentration seasons. It is considered that the size effects observed during the low-activity-concentration season may be due to factors related to metabolism and changes in food habit. This is the first long-term survey of 137Cs in wild boar inside the DRZ.

Genetic Population Structure of Wild Boars (Sus scrofa) in Fukushima Prefecture.

We aimed to reveal the dispersal and gene flow of the local wild boar (Sus scrofa) population and find their genetic boundary in Fukushima Prefecture. After the nuclear incident in 2011, the land was considered a difficult-to-return zone, and the increase in the number of wild boars was pronounced. To provide an effective management strategy for the wild boar population, we used multiplexed inter-simple sequence repeat genotyping by sequencing (MIG-seq) and clarified the genetic structure of wild boars. We obtained 328 single-nucleotide polymorphisms from 179 samples. STRUCTURE analysis showed that the most likely number of population cluster was K = 2. Molecular analysis of variance showed significant genetic differences between groups of wild boars inhabiting in the east and west across the Abukuma River. The migration rate from the eastern population to the western population is higher than in the reverse case based on BayesAss analysis. Our study indicates that both the Abukuma River and anthropogenic urbanization along the river may affect the migration of wild boars and the population in western was established mainly by the migration from other neighboring prefectures.

Molecular mechanisms of selenium tolerance and hyperaccumulation in Stanleya pinnata.

The molecular mechanisms responsible for selenium (Se) tolerance and hyperaccumulation were studied in the Se hyperaccumulator Stanleya pinnata (Brassicaceae) by comparing it with the related secondary Se accumulator Stanleya albescens using a combination of physiological, structural, genomic, and biochemical approaches. S. pinnata accumulated 3.6-fold more Se and was tolerant to 20 microm selenate, while S. albescens suffered reduced growth, chlorosis and necrosis, impaired photosynthesis, and high levels of reactive oxygen species. Levels of ascorbic acid, glutathione, total sulfur, and nonprotein thiols were higher in S. pinnata, suggesting that Se tolerance may in part be due to increased antioxidants and up-regulated sulfur assimilation. S. pinnata had higher selenocysteine methyltransferase protein levels and, judged from liquid chromatography-mass spectrometry, mainly accumulated the free amino acid methylselenocysteine, while S. albescens accumulated mainly the free amino acid selenocystathionine. S. albescens leaf x-ray absorption near-edge structure scans mainly detected a carbon-Se-carbon compound (presumably selenocystathionine) in addition to some selenocysteine and selenate. Thus, S. albescens may accumulate more toxic forms of Se in its leaves than S. pinnata. The species also showed different leaf Se sequestration patterns: while S. albescens showed a diffuse pattern, S. pinnata sequestered Se in localized epidermal cell clusters along leaf margins and tips, concentrated inside of epidermal cells. Transcript analyses of S. pinnata showed a constitutively higher expression of genes involved in sulfur assimilation, antioxidant activities, defense, and response to (methyl)jasmonic acid, salicylic acid, or ethylene. The levels of some of these hormones were constitutively elevated in S. pinnata compared with S. albescens, and leaf Se accumulation was slightly enhanced in both species when these hormones were supplied. Thus, defense-related phytohormones may play an important signaling role in the Se hyperaccumulation of S. pinnata, perhaps by constitutively up-regulating sulfur/Se assimilation followed by methylation of selenocysteine and the targeted sequestration of methylselenocysteine.

Expression and functions of myo-inositol monophosphatase family genes in seed development of Arabidopsis.

Myo-inositol monophosphatase (IMP) catalyzes the dephosphorylation of myo-inositol 3-phosphate in the last step of myo-inositol biosynthesis. IMP is also important in phosphate metabolism and is required for the biosynthesis of cell wall polysaccharides, phytic acid, and phosphatidylinositol. In Arabidopsis, IMP is encoded by VTC4. There are, however, two additional IMP candidate genes, IMPL1 and IMPL2, which have not yet been elucidated. In our genetic studies of Arabidopsis IMP genes, only the loss-of-function mutant impl2 showed embryonic lethality at the globular stage. All IMP genes were expressed in a similar manner both in the vegetative and reproductive organs. In developing seeds, expression of IMP genes was not coupled with the expression of the genes encoding myo-inositol phosphate synthases, which supply the substrate for IMPs in the de novo synthesis pathway. Instead, expression of IMP genes was correlated with expression of the gene for myo-inositol polyphosphate 1-phosphatase (SAL1), which is involved in the myo-inositol salvage pathway, suggesting a possible salvage pathway role in seed development. Moreover, the partial rescue of the impl2 phenotype by histidine application implies that IMPL2 is also involved in histidine biosynthesis during embryo development.

Untangling radiocesium dynamics of forest-stream ecosystems: A review of Fukushima studies in the decade after the accident.

Forest-stream ecosystems are widespread and biodiverse terrestrial landscapes with physical and social connections to downstream human activities. After radiocesium is introduced into these ecosystems, various material flows cause its accumulation or dispersal. We review studies conducted in the decade after the Fukushima nuclear accident to clarify the mechanisms of radiocesium transfer within ecosystems and to downstream areas through biological, hydrological, and geomorphological processes. After its introduction, radiocesium is heavily deposited in the organic soil layer, leading to persistent circulation due to biological activities in soils. Some radiocesium in soils, litter, and organisms is transported to stream ecosystems, forming contamination spots in depositional habitats. While reservoir dams function as effective traps, radiocesium leaching from sediments is a continual phenomenon causing re-contamination downstream. Integration of data regarding radiocesium dynamics and contamination sites, as proposed here, is essential for contamination management in societies depending on nuclear power to address the climate crisis.

Genetic Diversity of Invasive Spartina alterniflora Loisel. (Poaceae) Introduced Unintentionally Into Japan and Its Invasion Pathway.

Among invasive species, aquatic plants pose serious threats to local biodiversity and ecosystem functions. Spartina alterniflora Loisel. (Poaceae), native to the eastern United States, was introduced unintentionally into Japan (Aichi and Kumamoto Prefectures) at around 2010. This invasive species could easily and rapidly spread to estuarine areas of Japan via vigorous trade and transport, making the prediction of its future invasion necessary. Here, the distribution and structure of the genetic variation of S. alterniflora in Japan were examined using chloroplast DNA (cpDNA) and microsatellite genotyping analyses for clarifying its invasion route and process. According to the cpDNA analysis, S. alterniflora populations in Japan had a single haplotype (haplotype C4) that is the most dominant genotype around the Florida Peninsula, the region of its origin, and is also widely found in the introduced populations in the East Asia. Microsatellite analysis also showed a loss of genetic diversity in Japanese S. alterniflora populations (allelic richness (A R) = 1.20-1.39) compared with that in its native region (A R = 4.58-4.59), suggesting a founder effect on S. alterniflora that might have occurred after invasion of the species into Japan. The principal coordinate analysis and The STRUCTURE analysis indicated that no gene mixing among Japanese local populations (Aichi, northern and southern Kumamoto) was observed, indicating that Spartina invasion occurred independently into these regions. Among the three regions, trading between the ports of northern Kumamoto and the U.S. was obviously lower than trading with China. We concluded that invasive S. alterniflora might have independently invaded Japan at different times through an East Asia route, particularly via China (i.e., secondary introduction). Therefore, it is important to strengthen the quarantine control on the importation of commodities, especially of transport vehicles at potential donor spots (i.e., border control/border biosecurity system), and to share information networks on invasive species between each region/port for minimizing further risks of biological species such as Spartina.

The Arabidopsis sweetie mutant is affected in carbohydrate metabolism and defective in the control of growth, development and senescence.

SUMMARY: Sugars modulate many vital metabolic and developmental processes in plants, from seed germination to flowering, senescence and protection against diverse abiotic and biotic stresses. However, the exact mechanisms involved in morphogenesis, developmental signalling and stress tolerance remain largely unknown. Here we report the characterization of a novel Arabidopsis thaliana mutant, sweetie, with drastically altered morphogenesis, and a strongly modified carbohydrate metabolism leading to elevated levels of trehalose, trehalose-6-phosphate and starch. We additionally show that the disruption of SWEETIE causes significant growth and developmental alterations, such as severe dwarfism, lancet-shaped leaves, early senescence and flower sterility. Genes implicated in sugar metabolism, senescence, ethylene biosynthesis and abiotic stress were found to be upregulated in sweetie. Our physiological, biochemical, genetic and molecular data indicate that the mutation in sweetie was nuclear, single and recessive. The effects of metabolizable sugars and osmolytes on sweetie morphogenesis were distinct; in light, sweetie was hypersensitive to sucrose and glucose during vegetative growth and a partial phenotypic reversion took place in the presence of high sorbitol concentrations. However, SWEETIE encodes a protein that is unrelated to any known enzyme involved in sugar metabolism. We suggest that SWEETIE plays an important regulatory function that influences multiple metabolic, hormonal and stress-related pathways, leading to altered gene expression and pronounced changes in the accumulation of sugar, starch and ethylene.

Ethylene and salicylic acid control glutathione biosynthesis in ozone-exposed Arabidopsis thaliana.

Ozone produces reactive oxygen species and induces the synthesis of phytohormones, including ethylene and salicylic acid. These phytohormones act as signal molecules that enhance cell death in response to ozone exposure. However, some studies have shown that ethylene and salicylic acid can instead decrease the magnitude of ozone-induced cell death. Therefore, we studied the defensive roles of ethylene and salicylic acid against ozone. Unlike the wild-type, Col-0, Arabidopsis mutants deficient in ethylene signaling (ein2) or salicylic acid biosynthesis (sid2) generated high levels of superoxide and exhibited visible leaf injury, indicating that ethylene and salicylic acid can reduce ozone damage. Macroarray analysis suggested that the ethylene and salicylic acid defects influenced glutathione (GSH) metabolism. Increases in the reduced form of GSH occurred in Col-0 6 h after ozone exposure, but little GSH was detected in ein2 and sid2 mutants, suggesting that GSH levels were affected by ethylene or salicylic acid signaling. We performed gene expression analysis by real-time polymerase chain reaction using genes involved in GSH metabolism. Induction of gamma-glutamylcysteine synthetase (GSH1), glutathione synthetase (GSH2), and glutathione reductase 1 (GR1) expression occurred normally in Col-0, but at much lower levels in ein2 and sid2. Enzymatic activities of GSH1 and GSH2 in ein2 and sid2 were significantly lower than in Col-0. Moreover, ozone-induced leaf damage observed in ein2 and sid2 was mitigated by artificial elevation of GSH content. Our results suggest that ethylene and salicylic acid protect against ozone-induced leaf injury by increasing de novo biosynthesis of GSH.

DEAR1, a transcriptional repressor of DREB protein that mediates plant defense and freezing stress responses in Arabidopsis.

Plants have evolved intricate mechanisms to respond and adapt to a wide variety of biotic and abiotic stresses in their environment. The Arabidopsis DEAR1 (DREB and EAR motif protein 1; At3g50260) gene encodes a protein containing significant homology to the DREB1/CBF (dehydration-responsive element binding protein 1/C-repeat binding factor) domain and the EAR (ethylene response factor-associated amphiphilic repression) motif. We show here that DEAR1 mRNA accumulates in response to both pathogen infection and cold treatment. Transgenic Arabidopsis overexpressing DEAR1 (DEAR1ox) showed a dwarf phenotype and lesion-like cell death, together with constitutive expression of PR genes and accumulation of salicylic acid. DEAR1ox also showed more limited P. syringae pathogen growth compared to wild-type, consistent with an activated defense phenotype. In addition, transient expression experiments revealed that the DEAR1 protein represses DRE/CRT (dehydration-responsive element/C-repeat)-dependent transcription, which is regulated by low temperature. Furthermore, the induction of DREB1/CBF family genes by cold treatment was suppressed in DEAR1ox, leading to a reduction in freezing tolerance. These results suggest that DEAR1 has an upstream regulatory role in mediating crosstalk between signaling pathways for biotic and abiotic stress responses.

Comparison of 0.1 M Stable CsCl and 1 M NH4NO3 as an Extraction Reagent to Evaluate Cs-137 Mobility in Soils.

To evaluate the mobility and bioavailability of 137Cs in soils, we compared the extraction of 137Cs with stable Cs and ammonium solutions from 137Cs-contaminated minerals and soils. The extraction yields of 137Cs with stable Cs were significantly lower than those with ammonium for minerals with frayed edge sites, but such differences were not observed for minerals without frayed edge sites. The amount of 137Cs extracted with stable Cs from soils was lower than, or equal to, that extracted with ammonium. The above results suggest that stable Cs extracted the 137Cs from easily accessible sites. Plant available 137Cs was assessed using Kochia (Bassia scoparia) cultivated in pots of contaminated soils, and compared with soil parameters including extractable 137Cs and K, and radiocesium intercept potential. The 137Cs/K ratio extracted with stable Cs solution was found to be a potential index for evaluation of the easily mobile and bioavailable fraction of 137Cs in soil.

Isolation of O3-response genes from Arabidopsis thaliana using cDNA macroarray.

Nylon membrane-based cDNA macroarrays are a widely available alternative to cDNA microarrays for the collection of large-scale gene expression data. cDNA macroarrays are used in many areas of molecular biology research for applications ranging from gene discovery to gene expression profiling. Although degree of location of DNA spot in cDNA macroarray is lower than that in cDNA microarray, it can be used to detect expression of a large number of genes because it uses radiolabeled cDNA as a probe. Thus, cDNA macroarray technology can be applied to obtain the gene expression profile in organs that show wide variety in mRNA expression, such as meristems in plant species and brain tissue. To carry out hybridization experiments with a cDNA macroarray, I describe here how to prepare macroarray filters on a small or large scale, as well as how to analyze macroarray experiments and determine the statistical significance of the gene expression data obtained.

Transcriptome analyses give insights into selenium-stress responses and selenium tolerance mechanisms in Arabidopsis.

Selenate is chemically similar to sulfate and can be taken up and assimilated by plants via the same transporters and enzymes. In contrast to many other organisms, selenium (Se) has not been shown to be essential for higher plants. In excess, Se is toxic and restricts development. Both Se deficiency and toxicity pose problems worldwide. To obtain better insights into the effects of Se on plant metabolism and into plant mechanisms involved in Se tolerance, the transcriptome of Arabidopsis plants grown with or without selenate was studied and Se-responsive genes identified. Roots and shoots exhibited different Se-related changes in gene regulation and metabolism. Many genes involved in sulfur (S) uptake and assimilation were upregulated. Accordingly, Se treatment enhanced sulfate levels in plants, but the quantity of organic S metabolites decreased. Transcripts regulating the synthesis and signaling of ethylene and jasmonic acid were also upregulated by Se. Arabidopsis mutants defective in ethylene or jasmonate response pathways exhibited reduced tolerance to Se, suggesting an important role for these two stress hormones in Se tolerance. Selenate upregulated a variety of transcripts that were also reportedly induced by salt and osmotic stress. Selenate appeared to repress plant development, as suggested by the downregulation of genes involved in cell wall synthesis and auxin-regulated proteins. The Se-responsive genes discovered in this study may help create plants that can better tolerate and accumulate Se, which may enhance the effectiveness of Se phytoremediation or serve as Se-fortified food.

Glycosylation of bisphenol A by freshwater microalgae.

The endocrine disruptor bisphenol A (BPA, 4,4'-isopropylidenediphenol) is used to manufacture polycarbonate plastic and epoxy resin linings of food and beverage cans, and the residues from these products are then sometimes discharged into rivers and lakes in waste leachates. However, the fate of BPA in the environment has not yet been thoroughly elucidated. Considering the effect of BPA on aquatic organisms, it is important that we estimate the concentration of BPA and its metabolites in the aquatic environment, but there are few data on the metabolites of BPA. Here, we focused on freshwater microalgae as organisms that contribute to the biodegradation or biotransformation of BPA in aquatic environments. When we added BPA to cultures of eight species of freshwater microalgae, a reduction in the concentration of BPA in the culture medium was observed in all cultures. BPA was metabolized to BPA glycosides by Pseudokirchneriella subcapitata, Scenedesmus acutus, Scenedesmus quadricauda, and Coelastrum reticulatum, and these metabolites were then released into the culture medium. The metabolite from P. subcapitata, S. acutus, and C. reticulatum was identified by FAB-MS and (1)H-NMR as bisphenol A-mono-O-beta-d-glucopyranoside (BPAGlc), and another metabolite, from S. quadricauda, was identified as bisphenol A-mono-O-beta-d-galactopyranoside (BPAGal). These results demonstrate that freshwater microalgae that inhabit universal environments can metabolize BPA to its glycosides. Because BPA glycosides accumulate in plants and algae, and may be digested to BPA by beta-glycosidase in animal intestines, more attention should be given to levels of BPA glycosides in the environment to estimate the ecological impact of discharged BPA.

O2- activates leaf injury, ethylene and salicylic acid synthesis, and the expression of O3-induced genes in O3-exposed tobacco.

O3 is the major component of photochemical oxidants and gives rise to visible injuries on plant leaves. In O3-exposed plants, O2- is produced before the formation of the injury, but the role that O2- plays in plant response to 03 exposure is still unknown. To clarify its role, we observed the behavior of plants during O3 exposure after pretreatment with tiron, which is an O2- scavenger. When tiron-pretreated tobacco cv. Bel W3 was exposed to O3, leaf damage was attenuated. In O3-exposed tobacco, tiron inhibited increases in the levels of ethylene and salicylic acid, which promote leaf injury. Tiron pretreatment also suppressed increases in the expression of O3-induced genes. These results suggest that O2- is involved in many plant responses induced by O3 exposure. Bel B, a tobacco cultivar that is genetically related to Bel W3, is reported to be more resistant to O3 than Bel W3, but the reason for this difference is unclear. We investigated the differences between the responses of Bel B and tiron-pretreated Bel W3 to O3 exposure, and we discuss the reasons for the resistance to O3 by comparing the phenotype of Bel B with that of tiron-pretreated Bel W3.