Kenji Akiyama

Monitoring the expression pattern of around 7,000 Arabidopsis genes under ABA treatments using a full-length cDNA microarray.

Full-length cDNAs are essential for functional analysis of plant genes. Recently, cDNA microarray analysis has been developed for quantitative analysis of global and simultaneous analysis of expression profiles. Microarray technology is a powerful tool for identifying genes induced by environmental stimuli or stress and for analyzing their expression profiles in response to environmental signals. We prepared an Arabidopsis full-length cDNA microarray containing around 7,000 independent full-length cDNA groups and analyzed the expression profiles of genes. The transcripts of 245, 54, 299 and 213 genes increased after abscisic acid (ABA), drought-, cold-, and salt-stress treatments, respectively, with inducibilities more than fivefold compared with those of control genes. The cDNA microarray analysis showed that many ABA-inducible genes were induced after drought- and high-salinity-stress treatments, and that there is more crosstalk between drought and ABA responses than between ABA and cold responses. Among the ABA-inducible genes identified, we identified 22 transcription factor genes, suggesting that many transcriptional regulatory mechanisms exist in the ABA signal transduction pathways. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s10142-002-0070-6 or from http://www.gsc.riken.go.jp/Plant/index.html.Abstract. Full-length cDNAs are essential for functional analysis of plant genes. Recently, cDNA microarray analysis has been developed for quantitative analysis of global and simultaneous analysis of expression profiles. Microarray technology is a powerful tool for identifying genes induced by environmental stimuli or stress and for analyzing their expression profiles in response to environmental signals. We prepared an Arabidopsis full-length cDNA microarray containing around 7,000 independent full-length cDNA groups and analyzed the expression profiles of genes. The transcripts of 245, 54, 299 and 213 genes increased after abscisic acid (ABA), drought-, cold-, and salt-stress treatments, respectively, with inducibilities more than fivefold compared with those of control genes. The cDNA microarray analysis showed that many ABA-inducible genes were induced after drought- and high-salinity-stress treatments, and that there is more crosstalk between drought and ABA responses than between ABA and cold responses. Among the ABA-inducible genes identified, we identified 22 transcription factor genes, suggesting that many transcriptional regulatory mechanisms exist in the ABA signal transduction pathways. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s10142-002-0070-6 or from http://www.gsc.riken.go.jp/Plant/index.html.

Increased Expression of Intercellular Adhesion Molecule-1 (ICAM-1) in Diabetic Rat Glomeruli: Glomerular Hyperfiltration Is a Potential Mechanism of ICAM-1 Upregulation

Mononuclear cells, including monocytes/macrophages and T-cells, are considered to be involved in the progression of diabetic nephropathy, although the mechanism of their recruitment into diabetic glomeruli is unclear. The intercellular adhesion molecule-1 (ICAM-1) promotes the infiltration of leukocytes into atherosclerotic lesions as well as inflammatory tissues. In the present study, we investigated the expression of ICAM-1 in the glomeruli of streptozotocin-induced diabetic rats. The expression of ICAM-1 was increased significantly during the early stage of diabetes. The number of mononuclear cells, primarily monocytes/macrophages and lymphocytes, was significantly increased in diabetic glomeruli. Mononuclear cell infiltration into diabetic glomeruli was prevented by anti-ICAM-1 monoclonal antibody. Insulin treatment decreased ICAM-1 expression and mononuclear cell infiltration. The ICAM-1 expression on cultured human umbilical vein endothelial cells was not induced under high glucose culture conditions. Glomerular hyperfiltration is a characteristic change in the early stage of diabetic nephropathy. Treatment with aldose reductase inhibitor, which prevented glomerular hyperfiltration without changes in blood glucose levels, decreased ICAM-1 expression and mononuclear cell infiltration. Moreover, we examined the ICAM-1 expression in the glomeruli of the 5/6 nephrectomized rat, which is a model for glomerular hyperfiltration without hyperglycemia. The ICAM-1 expression and infiltration of mononuclear cells was significantly increased in the glomeruli of 5/6 nephrectomized rats. We conclude that ICAM-1 is upregulated and promotes the recruitment of mononuclear cells in diabetic glomeruli. Moreover, glomerular hyperfiltration that occurs in the early stage of diabetic glomeruli may be one of the potential mechanisms of ICAM-1 upregulation in diabetic nephropathy.

Widely Targeted Metabolomics Based on Large-Scale MS/MS Data for Elucidating Metabolite Accumulation Patterns in Plants

Metabolomics is an ‘omics’ approach that aims to analyze all metabolites in a biological sample comprehensively. The detailed metabolite profiling of thousands of plant samples has great potential for directly elucidating plant metabolic processes. However, both a comprehensive analysis and a high throughput are difficult to achieve at the same time due to the wide diversity of metabolites in plants. Here, we have established a novel and practical metabolomics methodology for quantifying hundreds of targeted metabolites in a high-throughput manner. Multiple reaction monitoring (MRM) using tandem quadrupole mass spectrometry (TQMS), which monitors both the specific precursor ions and product ions of each metabolite, is a standard technique in targeted metabolomics, as it enables high sensitivity, reproducibility and a broad dynamic range. In this study, we optimized the MRM conditions for specific compounds by performing automated flow injection analyses with TQMS. Based on a total of 61,920 spectra for 860 authentic compounds, the MRM conditions of 497 compounds were successfully optimized. These were applied to high-throughput automated analysis of biological samples using TQMS coupled with ultra performance liquid chromatography (UPLC). By this analysis, approximately 100 metabolites were quantified in each of 14 plant accessions from Brassicaceae, Gramineae and Fabaceae. A hierarchical cluster analysis based on the metabolite accumulation patterns clearly showed differences among the plant families, and family-specific metabolites could be predicted using a batch-learning self-organizing map analysis. Thus, the automated widely targeted metabolomics approach established here should pave the way for large-scale metabolite profiling and comparative metabolomics.

AtMetExpress development: a phytochemical atlas of Arabidopsis development.

Plants possess many metabolic genes for the production of a wide variety of phytochemicals in a tissue-specific manner. However, the metabolic systems behind the diversity and tissue-dependent regulation still remain unknown due to incomplete characterization of phytochemicals produced in a single plant species. Thus, having a metabolome dataset in addition to the genome and transcriptome information resources would enrich our knowledge of plant secondary metabolism. Here we analyzed phytochemical accumulation during development of the model plant Arabidopsis (Arabidopsis thaliana) using liquid chromatography-mass spectrometry in samples covering many growth stages and organs. We also obtained tandem mass spectrometry spectral tags of many metabolites as a resource for elucidation of metabolite structure. These are part of the AtMetExpress metabolite accumulation atlas. Based on the dataset, we detected 1,589 metabolite signals from which the structures of 167 metabolites were elucidated. The integrated analyses with transcriptome data demonstrated that Arabidopsis produces various phytochemicals in a highly tissue-specific manner, which often accompanies the expression of key biosynthesis-related genes. We also found that a set of biosynthesis-related genes is coordinately expressed among the tissues. These data suggested that the simple mode of regulation, transcript to metabolite, is an origin of the dynamics and diversity of plant secondary metabolism.

Monitoring the expression profiles of genes induced by hyperosmotic, high salinity, and oxidative stress and abscisic acid treatment in Arabidopsis cell culture using a full-length cDNA microarray.

Transcriptional regulation in response to hyperosmotic, high-salinity and oxidative stress, and abscisic acid (ABA) treatment in Arabidopsis suspension-cultured cell line T87 was investigated with a cDNA microarray containing 7000 independent full-length Arabidopsis cDNAs. The transcripts of 102, 11, 84 and 73 genes were increased more than 5-fold within 5h after treatment with 0.5M mannitol, 0.1M NaCl, 50μM ABA and 10mM H2O2, respectively. On the other hand, the transcripts of 44, 57, 25 and 34 genes were down-regulated to less than one-third within 5h after treatment with 0.5M mannitol, 0.1M NaCl, 50μM ABA and 10mM H2O2, respectively. Venn diagram analysis revealed 11 genes were induced significantly by mannitol, NaCl, and ABA, indicating crosstalk among these signaling pathways. Comparison of the genes induced by each stress revealed that 32%, 17% and 33% of mannitol-, NaCl- and ABA-inducible genes were also induced by H2O2, indicating the crosstalk between the signaling pathways for osmotic stress and oxidative stress. Although the expression profiles revealed that the T87 cells had most of the regulatory systems seen in Arabidopsis seedlings, the T87 cells did not have one of ABA-dependent signaling pathways.

Monitoring expression profiles of Arabidopsis genes during cold acclimation and deacclimation using DNA microarrays

A comparative analysis of gene expression profiles during cold acclimation and deacclimation is necessary to elucidate the molecular mechanisms of cold stress responses in higher plants. We analyzed gene expression profiles in the process of cold acclimation and deacclimation (recovery from cold stress) using two microarray systems, the 7K RAFL cDNA microarray and the Agilent 22K oligonucleotide array. By both microarray analyses, we identified 292 genes up-regulated and 320 genes down-regulated during deacclimation, and 445 cold up-regulated genes and 341 cold down-regulated genes during cold acclimation. Many genes up-regulated during deacclimation were found to be down-regulated during cold acclimation, and vice versa. The genes up-regulated during deacclimation were classified into (1) regulatory proteins involved in further regulation of signal transduction and gene expression and (2) functional proteins involved in the recovery process from cold-stress-induced damages and plant growth. We also applied expression profiling studies to identify the key genes involved in the biosynthesis of carbohydrates and amino acids that are known to play important roles in cold acclimation. We compared genes that are regulated during deacclimation with those regulated during rehydration after dehydration to discuss the similarity and difference of each recovery process.

RIKEN tandem mass spectral database (ReSpect) for phytochemicals: a plant-specific MS/MS-based data resource and database.

The fragment pattern analysis of tandem mass spectrometry (MS/MS) has long been used for the structural characterization of metabolites. The construction of a plant-specific MS/MS data resource and database will enable complex phytochemical structures to be narrowed down to candidate structures. Therefore, a web-based database of MS/MS data pertaining to phytochemicals was developed and named ReSpect (RIKEN tandem mass spectral database). Of the 3595 metabolites in ReSpect, 76% were derived from 163 literature reports, whereas the rest was obtained from authentic standards. As a main web application of ReSpect, a fragment search was established based on only the m/z values of query data and records. The confidence levels of the annotations were managed using the MS/MS fragmentation association rule, which is an algorithm for discovering common fragmentations in MS/MS data. Using this data resource and database, a case study was conducted for the annotation of untargeted MS/MS data that were selected after quantitative trait locus analysis of the accessions (Gifu and Miyakojima) of a model legume Lotus japonicus. In the case study, unknown metabolites were successfully narrowed down to putative structures in the website.

Increased expression of selectins in kidneys of patients with diabetic nephropathy

Summary In diabetic nephropathy leukocytes, mainly composed of monocytes/macrophages, which accumulate in the glomeruli and the interstitium, play an important part in the progression of glomerulosclerosis. The infiltration of leukocytes into inflammatory tissues or atherosclerotic lesions is mediated by adhesion molecules, which are expressed on the vascular endothelial cells, although little is known about the mechanism of leukocyte infiltration into diabetic renal tissues. P- and E-selectin are leukocyte adhesion molecules, which are expressed on the vascular endothelial cells and promote the adhesion of leukocytes to the endothelium. We investigated the expression of P- and E-selectin in the kidney tissue of patients with diabetic nephropathy and compared it with that of patients with other glomerular diseases (minimal change nephrotic syndrome, membranous nephropathy, IgA nephropathy, mesangioproliferative glomerulonephritis, and lupus nephritis). Expression of P- and E-selectin were both significantly increased in the glomeruli and the interstitium of patients with diabetic nephropathy as compared with those with other glomerular diseases. P- and E-selectin were both expressed along the glomerular capillaries and the peritubular capillaries in the interstitium. Neither P- nor E-selectin were correlated with the number of infiltrated leukocytes in the glomeruli, however, interestingly the E-selectin expression on peritubular capillaries was correlated with the number of infiltrated CD14 positive cells in the interstitium. These results suggest that E-selectin may play a key role in leukocyte infiltration into the renal interstitium in patients with diabetic nephropathy. [Diabetologia (1998) 41: 185–192]

Sequencing and analysis of approximately 40,000 soybean cDNA clones from a full-length-enriched cDNA library.

A large collection of full-length cDNAs is essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. We obtained a total of 39 936 soybean cDNA clones (GMFL01 and GMFL02 clone sets) in a full-length-enriched cDNA library which was constructed from soybean plants that were grown under various developmental and environmental conditions. Sequencing from 5′ and 3′ ends of the clones generated 68 661 expressed sequence tags (ESTs). The EST sequences were clustered into 22 674 scaffolds involving 2580 full-length sequences. In addition, we sequenced 4712 full-length cDNAs. After removing overlaps, we obtained 6570 new full-length sequences of soybean cDNAs so far. Our data indicated that 87.7% of the soybean cDNA clones contain complete coding sequences in addition to 5′- and 3′-untranslated regions. All of the obtained data confirmed that our collection of soybean full-length cDNAs covers a wide variety of genes. Comparative analysis between the derived sequences from soybean and Arabidopsis, rice or other legumes data revealed that some specific genes were involved in our collection and a large part of them could be annotated to unknown functions. A large set of soybean full-length cDNA clones reported in this study will serve as a useful resource for gene discovery from soybean and will also aid a precise annotation of the soybean genome.

Statistical Indices for Simultaneous Large-Scale Metabolite Detections for a Single NMR Spectrum

NMR-based metabolomics has become a practical and analytical methodology for discovering novel genes, biomarkers, metabolic phenotypes, and dynamic cell behaviors in organisms. Recent developments in NMR-based metabolomics, however, have not concentrated on improvements of comprehensiveness in terms of simultaneous large-scale metabolite detections. To resolve this, we have devised and implemented a statistical index, the SpinAssign p-value, in NMR-based metabolomics for large-scale metabolite annotation and publicized this information. It enables simultaneous annotation of more than 200 candidate metabolites from the single (13)C-HSQC (heteronuclear single quantum coherence) NMR spectrum of a single sample of cell extract.