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α-Synuclein (α-Syn) binds to vesicle-associated membrane protein-binding protein B (VAPB) in the endoplasmic reticulum membrane. Recent studies have shown that α-Syn-immunoreactive Lewy pathology is characterized by membrane crowding, including vesicular structures. To elucidate the role of VAPB and vesicular structures in Parkinson's disease (PD) and in dementia with Lewy bodies (DLB), the relationships among VAPB, vesicular structures, and Lewy pathology were investigated by immunohistochemistry and immunoelectron microscopy in 8 PD and 4 DLB autopsy cases. The proportions of VAPB-negative neurons in the substantia nigra in PD and in the temporal cortex in DLB were significantly higher than those in 5 controls. In PD, the incidence of α-Syn inclusions in VAPB-negative neurons was significantly higher (77.4%) than in VAPB-positive neurons (1.6%) in the substantia nigra. In DLB, the incidence of α-Syn inclusions in VAPB-negative neurons was also significantly higher (65.3%) than in VAPB-positive neurons (2.8%) in the temporal cortex. Immunoelectron microscopy revealed that α-Syn and VAPB were localized to filamentous structures of Lewy bodies (LBs). However, only a few vesicular structures labeled with anti-α-Syn were observed within LBs. These findings suggest that reduction of VAPB is involved in the disease processes of PD and DLB, although vesicular structures may not directly contribute to the formation of LBs.

Todo-matsu (Abies sachalinensis) is one of the most important forestry species in Hokkaido, Japan and is distributed from near sea level to the alpine zone. Due to its wide spatial distribution, the species adapts to its environment, displaying phenotypes of ecological relevance. In order to identify candidate genes under natural selection, we collected the transcriptome from the female and male flower, leaf and inner bark. De novo assembly with 34.7 Gb of sequencing reads produced 158,542 transcripts from 69,618 loci, whose estimated coverage reached 95.6% of conserved eukaryotic genes. Homology searches against publicly available databases identified 134,190 (84.6%) transcripts with at least one hit. In total, 28,944 simple sequence repeats (SSRs) and 80,758 single nucleotide variants (SNVs) were detected from 23,570 (14.9%) and 25,366 (16.0%) transcripts, which were valuable for use in genetic analysis of the species. All the annotations were included in a relational database, TodoFirGene, which provides an interface for various queries and homology search, and can be accessed at http://plantomics.mind.meiji.ac.jp/todomatsu/. This database hosts not only the A. sachalinensis transcriptome but also links to the proteomes of 13 other species, allowing a comparative genomic study of plant species.

Japanese pear (Pyrus pyrifolia) is a major fruit tree in the family Rosaceae and is bred for fruit production. To promote the development of breeding strategies and molecular research for Japanese pear, we sequenced the transcripts of Japanese pear variety ‘Hosui’. To exhaustively collect information of total gene expression, RNA samples from various organs and stages of Japanese pear were sequenced by three technologies, single-molecule real-time (SMRT) sequencing, 454 pyrosequencing, and Sanger sequencing. Using all those reads, we determined comprehensive reference sequences of Japanese pear. Then, their protein sequences were predicted, and biological functional annotations were assigned. Finally, we developed a web database, TRANSNAP (http://plantomics.mind.meiji.ac.jp/nashi), which is the first web resource of Japanese pear omics information. This database provides highly reliable information via a user-friendly web interface: the reference sequences, gene functional annotations, and gene expression profiles from microarray experiments. In addition, based on sequence comparisons among Japanese, Chinese and European pears, similar protein sequences among the pears and species-specific proteins in Japanese pear can be quickly and efficiently identified. TRANSNAP will aid molecular research and breeding in Japanese pear, and its information is available for comparative analysis among other pear species and families.

Watercore is a physiological disorder of Japanese pear fruits, in which a watery translucent area appears in the flesh of fruits on the tree. The disorder leads to the loss of commercial value, and resistance to watercore is one of the most important features to be studied in the breeding of Japanese pear. Even in susceptible cultivars such as ‘Hosui’ (syn. ‘Housui’), the degree of watercore development varies markedly from year to year, which makes it difficult to analyze the characteristics of this phenomenon prior to visible watercore development. We used two F1 sib pairs from ‘Hosui’ × ‘Hougetsu’ and ‘Akiakari’ × ‘Taihaku’ crosses. Each pair contains a susceptible sib and a resistant sib. In these pairs, susceptible sibs developed severe watercore in all the fruits examined in each year, whereas no watercore was found even in overmature fruits of resistant sibs. To search for genes related to watercore, we used a custom pear microarray and studied differential gene expression in susceptible and resistant sibs prior to visible watercore development. We identified 115 differentially expressed genes including sugar metabolism–, hormone–, and cell wall–related genes. Some of these genes were differentially expressed only in immature fruits prior to watercore development. These genes would be useful as molecular markers to predict watercore at early developmental stages or in pear breeding programs.

Simple sequence repeat (SSR) markers are highly informative and widely used in genetic studies and plant breeding. Sporobolus virginicus is a halophytic turf grass that shows a high tolerance to salinity of up to 1.5 M NaCl. In the present study, we developed 148,411 SSR markers on 48,512 transcriptome contigs derived from RNA sequencing data. Of 33 randomly selected SSR markers, 23 (69.7%) produced clean amplification products, and an average of 1.25 alleles per marker was detected in S. virginicus genotypes. These markers were also examined by detecting polymorphisms across 19 different genera/species/genotypes in Poaceae, resulting in a high percentage (40%) of transferability. The sequencing of amplified products from these genera/species/genotypes revealed a high level of sequence similarity; however, substitutions, deletions, and insertions were detected not only in the objective SSRs but also in their flanking sequences. This is the first study on SSR marker development from S. virginicus. These SSR markers are useful for mapping the genes in and the breeding of S. virginicus and also for comparative genomics across genera/species/genotypes in Poaceae.

Analysis of a gene expression network (GEN), which is constructed based on similarity of gene expression profiles, is a widely used approach to gain clues for new biological insights. The recent abundant availability of transcriptome data in public databases is enabling GEN analysis under various experimental conditions, and even comparative GEN analysis across species. To provide a platform to gain biological insights from public transcriptome data, valuable databases have been created and maintained. This chapter introduces the web database OryzaExpress, providing omics information on Oryza sativa (rice). The integrated database Plant Omics Data Center, supporting a wide variety of plant species, is also described to compare omics information among multiple plant species.

Analysis of a gene expression network (GEN), which is constructed based on similarity of gene expression profiles, is a widely used approach to gain clues for new biological insights. The recent abundant availability of transcriptome data in public databases is enabling GEN analysis under various experimental conditions, and even comparative GEN analysis across species. To provide a platform to gain biological insights from public transcriptome data, valuable databases have been created and maintained. This chapter introduces the web database OryzaExpress, providing omics information on Oryza sativa (rice). The integrated database Plant Omics Data Center, supporting a wide variety of plant species, is also described to compare omics information among multiple plant species.

“Fairy rings” resulting from fungus-stimulated plant growth occur all over the world. In 2010, 2-azahypoxanthine (AHX) from a fungus Lepista sordida was identified as the “fairy” that stimulates plant growth. Furthermore, 2-aza-8-oxohypoxanthine (AOH) was isolated as a common metabolite of AHX in plants, and the endogenous existence of AHX and AOH in plants was proved. The structure of AHX allowed us to hypothesize that AHX was derived from 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Thus, we performed a feeding experiment that supplied AICAR to L. sordida. Consumption of AICAR and accumulation of AHX were observed after feeding. The mycelia extract had enzymatic activity of adenine/5-aminoimidazole-4-carboxamide phosphoribosyltransferase (APRT). APRT gene of L. sordida revealed its structural characteristics in homology modeling and showed transcriptional enhancement after feeding. These results support that AHX was synthesized from AICAR and AHX biosynthesis was transcriptionally controlled by AICAR, indicating the presence of novel purine metabolic pathway in L. sordida.

Steroidal glycoalkaloids (SGAs) are cholesterol-derived specialized metabolites produced in species of the Solanaceae. Here, we report that a group of jasmonate-responsive transcription factors of the ETHYLENE RESPONSE FACTOR (ERF) family (JREs) are close homologs of alkaloid regulators in Cathranthus roseus and tobacco, and regulate production of SGAs in tomato. In transgenic tomato, overexpression and dominant suppression of JRE genes caused drastic changes in SGA accumulation and in the expression of genes for metabolic enzymes involved in the multistep pathway leading to SGA biosynthesis, including the upstream mevalonate pathway. Transactivation and DNA-protein binding assays demonstrate that JRE4 activates the transcription of SGA biosynthetic genes by binding to GCC box-like elements in their promoters. These JRE-binding elements occur at significantly higher frequencies in proximal promoter regions of the genes regulated by JRE genes, supporting the conclusion that JREs mediate transcriptional co-ordination of a series of metabolic genes involved in SGA biosynthesis.

We present a high-resolution map of genomic transformation-competent artificial chromosome (TAC) clones extending over all Arabidopsis thaliana (Arabidopsis) chromosomes. The Arabidopsis genomic TAC clones have been valuable genetic tools. Previously, we constructed an Arabidopsis genomic TAC library consisting of more than 10,000 TAC clones harboring large genomic DNA fragments extending over the whole Arabidopsis genome. Here, we determined 13,577 end sequences from 6987 Arabidopsis TAC clones and mapped 5937 TAC clones to precise locations, covering approximately 90% of the Arabidopsis chromosomes. We present the large-scale data set of TAC clones with high-resolution mapping information as a Java application tool, the Arabidopsis TAC Position Viewer, which provides ready-to-go transformable genomic DNA clones corresponding to certain loci on Arabidopsis chromosomes. The TAC clone resources will accelerate genomic DNA cloning, positional walking, complementation of mutants and DNA transformation for heterologous gene expression.

The number of cells and the rate of cell division were observed in diploid parthenogenetic mouse embryos during the course of blastocyst formation, and were compared with those in embryos developed from fertilized ova (fertilized embryos), in order to identify the stage at which the number of cells begins to differ and the cause of difference in cell number between parthenogenetic and fertilized embryos. The number of cells and the rate of cell division did not differ between parthenogenetic and fertilized embryos at the 8-cell and compacted morula stages. The numbers of inner-cell-mass and trophoblast cells did not differ between parthenogenetic and fertilized embryos at the early blastocyst stage, but were significantly fewer in parthenogenetic embryos (11.1 and 32.7) than in fertilized embryos (14.9 and 52.0) at the expanded blastocyst stage. The numbers of dead inner-cell-mass and trophoblast cells were significantly more in parthenogenetic embryos than in fertilized ones at the early and expanded blastocyst stages. At the early blastocyst stage, although no difference was observed between parthenogenetic and fertilized embryos in the rate of division of trophoblast cells, the rate of division of inner-cell-mass cells was significantly lower in parthenogenetic embryos (4.4%) than in fertilized ones (9.7%). There was no difference in the rate of cell division between parthenogenetic and fertilized embryos at the expanded blastocyst stage. From these results, it was inferred that the number of cells in diploid parthenogenetic embryos does not differ from fertilized embryos until blastocyst formation, but becomes fewer than in fertilized embryos because more cells are dying in parthenogenetic embryos during the expansion of blastocysts. A low rate of cell division in the early blastocyst stage is thought to be one reason for the fewer number of inner-cell-mass cells in parthenogenetic embryos at the expanded stage, in addition to the presence of a large number of dead cells.

For proper control of biological activity, some key genes are highly expressed in a particular spatiotemporal domain. Mining of such spatiotemporally expressed genes using large-scale gene expression data derived from a broad range of experimental sources facilitates our understanding of genome-scale functional gene networks. However, comprehensive information on spatiotemporally expressed genes is lacking in plants. To collect such information, we devised a new index, Δdmax, which is the maximum difference in relative gene expression levels between sample runs which are neighboring when sorted by the levels. Employing this index, we comprehensively evaluated transcripts using large-scale RNA sequencing (RNA-Seq) data stored in the Sequence Read Archive for eight plant species: Arabidopsis thaliana (Arabidopsis), Solanum lycopersicum (tomato), Solanum tuberosum (potato), Oryza sativa (rice), Sorghum bicolor (sorghum), Vitis vinifera (grape), Medicago truncatula (Medicago), and Glycine max (soybean). Based on the frequency distribution of the Δdmax values, approximately 70,000 transcripts showing 0.3 or larger Δdmax values were extracted for the eight species. Information on these genes including the Δdmax values, functional annotations, conservation among species, and experimental conditions where the genes show high expression levels is provided in a new database, CATchUP (http://plantomics.mind.meiji.ac.jp/CATchUP). The CATchUP database assists in identifying genes specifically expressed under particular conditions with powerful search functions and an intuitive graphical user interface.

Legumes, the third largest family of flowering plants, are composed of more than 18,000 species with diverse characteristics, and many of the species are of agronomic importance. They have long been the targets of breeding programs and, in this regard, genomics may provide the greatest potential for benefit. Legume genomics in two model plants, Lotus japonicus and Medicago truncatula, has advanced rapidly over the past few years (Young

Jasmonates are known as growth regulators, which have cyclopentanone or cyclopentenone ring, and synthesized through lipoxygenase pathway. Jasmonates are widely distributed in the plant kingdom and modulates wounding responses, disease responses, and anther development. Some kinds of jasmonates were shown to have specific effects on plants. 12-oxo-phytodienoic acid (OPDA), a precursor for jasmonic acid (JA) biosynthesis, promotes tendril coiling, and this effect is stronger than MeJA (Falkenstein E., et al., 1991). However, except for the fact, little is known about the OPDA specific function in various physiological events.

Advances in DNA cloning and sequencing technologies have allowed the performance of comprehensive analysis of genetic information in various flowering plants of biological and agronomical importance. Among them, Arabidopsis thaliana, a member of the Brassica family, was chosen as a plant most suitable for genomic sequencing (Goodman et al. 1995; Meinke et al. 1998), because the estimated genome size of 125–130Mb is the smallest among known higher plants and the content of repetitive sequences was assumed to be low. Its short life cycle (average 60 days) and prodigious seed production are the characteristics which make this small plant an ideal model organism in which to analyze metabolism, development, stress responses, and disease resistance in all the flowering plants