Details of a Researcher - Tomita, Masaru

Papers - Tomita, Masaru

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Polymer Entrapment in Polymerized Silicate for Preparing Highly Stable Capillary Coatings for CE and CE-MS

Monton, M., Tomita, M., Soga, T. and Ishihama, Y.

Analytical Chemistry 79 ( 20 ) 7838-7844 - 7844 2007

Research paper (scientific journal), Joint Work, ISSN 00032700

An easy-to-implement capillary coating strategy based on polymer entrapment in the network of polymerized silicate is described. In this manner, cationic polymers are tightiy fixed onto the inner wall of the capillary for electroosmotic flow control without necessitating complex surface modification chemistries. The resulting coated capillary exhibited good stability over a wide range of pH, good reproducibility, strong endurance in more than 300 electrophoretic runs, and tolerance of commonly employed organic solvent additives in CE. Applications in CE-MS analysis of biologically important anions as well as sample enrichment are shown. Additionally, it was used as a durable base for attachment of multiple layers of charged polymers on the wall, via electrostatic interaction with the preceding layer. Thus, two novel types of highly stable coated capillaries, one with anodic EOF and the other cathodic, were developed. © 2007 American Chemical Society.

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Phosphopeptide Enrichment by Aliphatic Hydroxy Acid-modified Metal Oxide Chromatography for Nano-LC-MS/MS in Proteomics Applications

Sugiyama, N., Masuda, T., Shinoda, K., Nakamura, A., Tomita, M. and Ishihama, Y.

Mol Cell Proteomics 6 ( 6 ) 1103-1109 - 1109 2007

Research paper (scientific journal), Joint Work, ISSN 15359476

We developed novel methods for phosphopeptide enrichment using aliphatic hydroxy acid-modified metal oxide chromatography (MOC). Titania and zirconia were successfully applied to enrich phosphopeptides with the aid of aliphatic hydroxy acids, such as lactic acid and β-hydroxypropanoic acid, to reduce the interaction between acidic non-phosphopeptides and the metal oxides. These methods removed the vast majority of non-phosphopeptides from phosphoprotein standard digests, and large numbers of phosphopeptides could be readily identified. The methods were coupled with nano-LC-MS/MS systems without difficulty. Recovery of phosphopeptides in MOC varied greatly from peptide to peptide, ranging from a few percent to 100%, and the average was almost 50%. Repeatability and linearity were satisfactory. In an examination of the cytoplasmic fraction of HeLa cells, more than 1000 phosphopeptides were identified using lactic acid-modified titania MOC and β-hydroxypropanoic acid-modified zirconia MOC, respectively. The overlap between phosphopeptides enriched by these two methods was 40%, and the combined results provided 1646 unique phosphopeptides. To our knowledge, this is the first successful application of a single MOC-based approach to phosphopeptide enrichment from complex biological samples such as cell lysates. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Statistical Analysis of Gene Expression in Innate Immune Responses: Dynamic Interactions between MicroRNA and Signaling Molecules

Piras, V., Selvarajoo, K., Fujikawa, N., Choi, S., Tomita, M., Giuliani, A. and Tsuchiya, M.

Genomics and Informatics 5 107-112 2007

Research paper (scientific journal), Joint Work

Multiple high throughput analyses monitor the response of E. coli to perturbations

Ishii, N., Nakahigashi, K., Baba, T., Robert, M., Soga, T., Kanai, A., Hirasawa, T., Naba, M., Hirai, K., Hoque, A., Yee, Ho, Pei., Kakazu, Y., Sugawara, K., Igarashi, S., Harada, S., Masuda, T., Sugiyama, N., Togashi, T., Hasegawa, M., Takai, T., Yugi, K., Arakawa, K., Iwata, N., Toya, Y., Nakayama, Y., Nishioka, T., Shimizu, K., Mori, H. and Tomita, M.

Science 316 ( 5824 ) 593-597 - 597 2007

Research paper (scientific journal), Joint Work, ISSN 00368075

Analysis of cellular components at multiple levels of biological information can provide valuable functional insights. We performed multiple high-throughput measurements to study the response of Escherichia coli cells to genetic and environmental perturbations. Analysis of metabolic enzyme gene disruptants revealed unexpectedly small changes in messenger RNA and proteins for most disruptants. Overall, metabolite levels were also stable, reflecting the rerouting of fluxes in the metabolic network. In contrast, E. coli actively regulated enzyme levels to maintain a stable metabolic state in response to changes in growth rate. E. coli thus seems to use complementary strategies that result in a metabolic network robust against perturbations.

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Network scaling invariants help to elucidate basic topological principles of proteins

Krishnan, A., Giuliani, A., Zbilut, J.P. and Tomita, M.

Journal of Proteome Research 6 ( 10 ) 3924-3934 - 3934 2007

Research paper (scientific journal), Joint Work, ISSN 15353893

The structural architecture of proteins continues to be an area of active research. Despite the difference in models dealing with the way proteins fold into their tertiary structures, it is recognized that small regions of proteins tend to fold independently and are then stabilized by interactions between these distinct subunits. However, there are a number of different definitions of what comprises an independent subunit. In the belief that an unequivocal definition of a domain must be based on the most fundamental property of protein 3D structure, namely, the adjacency matrix of inter-residues contact, we adopt a network representation of the protein. In this work, we used a well-established, global method for identifying modules in networks, without any specific reference to the kind of network being analyzed. The algorithm converges toward the maximization of the modularity of the given protein network and, in doing so, allows the representation of the residues of the protein in terms of their intramodule degree, z, and participation coefficient, P. We demonstrate that the labeling of residues in terms of these invariants allows for information-rich representations of the studied proteins as well as to sketch a new way to link sequence, structure, and the dynamical properties of proteins. We discovered a strong invariant character of protein molecules in terms of P/z characterization, pointing to a common topological design of all protein structures. This invariant representation, applied to different protein systems, enabled us to identify the possible functional role of high P/z residues during the folding process. Additionally, we observe a hierarchical behavior of protein structural organization that provides a sequence-secondary-tertiary structure link. The discovery of similar and repeatable scaling laws at different level of definitions going from hydrophobicity patterning along the sequence up to the size of an autonomous folding unit (AFU) and general contact distribution of the entire molecule suggest a hierarchical-like behavior of protein architecture. This implies the possibility to select different privileged scales of observation for deriving useful information on protein systems. © 2007 American Chemical Society.

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Inferring rules of Escherichia coli translational efficiency using an artificial neural network

Mori, K., Saito, R., Kikuchi, S. and Tomita, M.

BioSystems 90 ( 2 ) 414-420 - 420 2007

Research paper (scientific journal), Joint Work, ISSN 03032647

Although the machinery for translation initiation in Escherichia coli is very complicated, the translational efficiency has been reported to be predictable from upstream oligonucleotide sequences. Conventional models have difficulties in their generalization ability and prediction nonlinearity and in their ability to deal with a variety of input attributions. To address these issues, we employed structural learning by artificial neural networks to infer general rules for translational efficiency. The correlation between translational activities measured by biological experiments and those predicted by our method in the test data was significant (r = 0.78), and our method uncovered underlying rules of translational activities and sequence patterns from the obtained skeleton structure. The significant rules for predicting translational efficiency were (1) G- and A-rich oligonucleotide sequences, resembling the Shine-Dalgarno sequence, at positions -10 to -7; (2) first base A in the initiation codon; (3) transport/binding or amino acid metabolism gene function; (4) high binding energy between mRNA and 16S rRNA at positions -15 to -5. An additional inferred novel rule was that C at position -1 increases translational efficiency. When our model was applied to the entire genomic sequence of E. coli, translational activities of genes for metabolism and translational were significantly high. © 2006 Elsevier Ireland Ltd. All rights reserved.

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Computational Methods for MicroRNA Target Prediction

Watanabe, Y., Tomita, M. and Kanai, A.

Methods in Enzymology 427 65-86 - 86 2007

Research paper (scientific journal), Joint Work, ISSN 00766879

The discovery of microRNAs (miRNAs) has introduced a new paradigm into gene regulatory systems. Large numbers of miRNAs have been identified in a wide range of species, and most of them are known to downregulate translation of messenger RNAs (mRNAs) via imperfect binding of the miRNA to a specific site or sites in the 3′ untranslated region (UTR) of the mRNA. Identification of genes targeted by miRNAs is widely believed to be an important step toward understanding the role of miRNAs in gene regulatory networks. As part of the effort to understand interactions between miRNAs and their targets, computational algorithms have been developed based on observed rules for features such as the degree of hybridization between the two RNA molecules. These in silico approaches provide important tools for miRNA target detection, and together with experimental validation, help to reveal regulated targets of miRNAs. Here, we summarize the knowledge that has been accumulated about the principles of target recognition by miRNAs and the currently available computational methodologies for prediction of miRNA target genes. © 2007 Elsevier Inc. All rights reserved.

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Selection effects on the positioning of genes and gene structures from the interplay of replication and transcription in bacterial genomes

Arakawa, K. and Tomita, M.

Evolutionary Bioinformatics 3 279-286 - 286 2007

Research paper (scientific journal), Joint Work

Bacterial chromosomes are partly shaped by the functional requirements for effi cient replication, which lead to strand bias as commonly characterized by the excess of guanines over cytosines in the leading strand. Gene structures are also highly organized within bacterial genomes as a result of such functional constraints, displaying characteristic positioning and structuring along the genome. Here we analyze the gene structures in completely sequenced bacterial chromosomes to observe the positional constraints on gene orientation, length, and codon usage with regard to the positions of replication origin and terminus. Selection on these gene features is different in regions surrounding the terminus of replication from the rest of the genome, but the selection could be either positive or negative depending on the species, and these positional effects are partly attributed to the A-T enrichment near the terminus. Characteristic gene structuring relative to the position of replication origin and terminus is commonly observed among most bacterial species with circular chromosomes, and therefore we argue that the highly organized gene positioning as well as the strand bias should be considered for genomics studies of bacteria.

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In silico screening of archaeal tRNA-encoding genes having multiple introns with bulge-helix-bulge splicing motifs

Sugahara, J., Yachie, N., Arakawa, K. and Tomita, M.

RNA 13 ( 5 ) 671-681 - 681 2007

Research paper (scientific journal), Joint Work, ISSN 13558382

In archaeal species, several transfer RNA genes have been reported to contain endogenous introns. Although most of the introns are located at anticodon loop regions between nucleotide positions 37 and 38, a number of introns at noncanonical sites and six cases of tRNA genes containing two introns have also been documented. However, these tRNA genes are often missed by tRNAscan-SE, the software most widely used for the annotation of tRNA genes. We previously developed SPLITS, a computational tool to identify tRNA genes containing one intron at a noncanonical position on the basis of its discriminative splicing motif, but the software was limited in the detection of tRNA genes with multiple introns at noncanonical sites. In this study, we initially updated the system as SPLITSX in order to correctly predict known tRNA genes as well as novel ones with multiple introns. By a comprehensive search for tRNA genes in 29 archaeal genomes using SPLITSX, we listed 43 novel candidates that contain introns at noncanonical sites. As a result, 15 contained two introns and three contained three introns within the respective putative tRNA genes. Moreover, the candidates completely complemented all the codons of two archaeal species of uncultured methanogenic archaeon, RC-I and Thermofilum pendens Hrk 5, with novel candidates that were not detectable by tRNAscan-SE alone. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 RNA Society.

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Inhibitory effect of phosphoenolpyruvate on glycolytic enzymes in Escherichia coli

Ogawa, T., Mori, H., Tomita, M. and Yoshino, M.

Research in Microbiology 158 ( 2 ) 159-163 - 163 2007

Research paper (scientific journal), Joint Work, ISSN 09232508

For analyzing the control of energy metabolism in Escherichia coli, we carried out kinetic analyses of glycolytic enzymes purified from the overexpressing clones of E. coli K12 W3110 that were constructed with the vector pCA24N. Phosphoenolpyruvate (PEP) acted as an effective inhibitor of enzymes of the preparatory phase in glycolysis. Glucokinase was potently inhibited by PEP in a competitive manner with respect to ATP: the Ki value for PEP was 0.1 mM. PEP further inhibited phosphoglucoisomerase to a lesser extent, and phosphofructokinase A and aldolase A with 10-fold the Ki values of glucokinase and phosphoglucoisomerase. Glucose is incorporated into E. coli through two pathways: the PTS (PEP-dependent phosphotransferase system) and the glucokinase reaction. PEP, a potent inhibitor of E. coli glucokinase, unlike most eukaryotic hexokinases, can act as a signal molecule controlling glucose uptake and glycolytic flux in cells. © 2006 Elsevier Masson SAS. All rights reserved.

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Simulation of developmental changes in action potentials with ventricular cell models

Itoh, H., Naito, Y. and Tomita, M.

Systems and Synthetic Biology 1 ( 1 ) 11-23 - 23 2007

Research paper (scientific journal), Joint Work, ISSN 18725325

During cardiomyocyte development, early embryonic ventricular cells show spontaneous activity that disappears at a later stage. Dramatic changes in action potential are mediated by developmental changes in individual ionic currents. Hence, reconstruction of the individual ionic currents into an integrated mathematical model would lead to a better understanding of cardiomyocyte development. To simulate the action potential of the rodent ventricular cell at three representative developmental stages, quantitative changes in the ionic currents, pumps, exchangers, and sarcoplasmic reticulum (SR) Ca2+ kinetics were represented as relative activities, which were multiplied by conductance or conversion factors for individual ionic systems. The simulated action potential of the early embryonic ventricular cell model exhibited spontaneous activity, which ceased in the simulated action potential of the late embryonic and neonatal ventricular cell models. The simulations with our models were able to reproduce action potentials that were consistent with the reported characteristics of the cells in vitro. The action potential of rodent ventricular cells at different developmental stages can be reproduced with common sets of mathematical equations by multiplying conductance or conversion factors for ionic currents, pumps, exchangers, and SR Ca 2+ kinetics by relative activities. © Springer Science + Business Media B.V. 2007.

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Visualization of three-way comparisons of omics data

Baran, R., Robert, M., Suematsu, M., Soga, T. and Tomita, M.

BMC Bioinformatics 8 72 2007

Research paper (scientific journal), Joint Work

Background: Density plot visualizations (also referred to as heat maps or color maps) are widely used in different fields including large-scale omics studies in biological sciences. However, the current color-codings limit the visualizations to single datasets or pairwise comparisons. Results: We propose a color-coding approach for the representation of three-way comparisons. The approach is based on the HSB (hue, saturation, brightness) color model. The three compared values are assigned specific hue values from the circular hue range (e.g. red, green, and blue). The hue value representing the three-way comparison is calculated according to the distribution of three compared values. If two of the values are identical and one is different, the resulting hue is set to the characteristic hue of the differing value. If all three compared values are different, the resulting hue is selected from a color gradient running between the hues of the two most distant values (as measured by the absolute value of their difference) according to the relative position of the third value between the two. The saturation of the color representing the three-way comparison reflects the amplitude (or extent) of the numerical difference between the two most distant values according to a scale of interest. The brightness is set to a maximum value by default but can be used to encode additional information about the three-way comparison. Conclusion: We propose a novel color-coding approach for intuitive visualization of three-way comparisons of omics data. © 2007 Baran et al; licensee BioMed Central Ltd.

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Alignment-Based Approach for Durable Data Storage into Living Organisms

Yachie, N., Sekiyama, K., Sugahara, J., Ohashi, Y. and Tomita, M.

Biotechnology Progress 23 ( 2 ) 501-505 - 505 2007

Research paper (scientific journal), Joint Work, ISSN 87567938

The practical realization of DNA data storage is a major scientific goal. Here we introduce a simple, flexible, and robust data storage and retrieval method based on sequence alignment of the genomic DNA of living organisms. Duplicated data encoded by different oligonucleotide sequences was inserted redundantly into multiple loci of the Bacillus subtilis genome. Multiple alignment of the bit data sequences decoded by B. subtilis genome sequences enabled the retrieval of stable and compact data without the need for template DNA, parity checks, or error-correcting algorithms. Combined with the computational simulation of data retrieval from mutated message DNA, a practical use of this alignment-based method is discussed. © 2007 American Chemical Society and American Institute of Chemical Engineers.

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Dynamic simulation of an in vitro multi-enzyme system

Ishii, N., Suga, Y., Hagiya, A., Watanabe, H., Mori, H., Yoshino, M, and Tomita, M.

FEBS Letters 581 ( 3 ) 413-420 - 420 2007

Research paper (scientific journal), Joint Work, ISSN 00145793

Parameters often are tuned with metabolite concentration time series data to build a dynamic model of metabolism. However, such tuning may reduce the extrapolation ability (generalization capability) of the model. In this study, we determined detailed kinetic parameters of three purified Escherichia coli glycolytic enzymes using the initial velocity method for individual enzymes; i.e., the parameters were determined independently from metabolite concentration time series data. The metabolite concentration time series calculated by the model using the parameters matched the experimental data obtained in an actual multi-enzyme system consisting of the three purified E. coli glycolytic enzymes. Thus, the results indicate that kinetic parameters can be determined without using an undesirable tuning process. © 2007 Federation of European Biochemical Societies.

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Comparative analysis of cis-encoded antisense RNAs in eukaryotes

Numata, K., Okada, Y., Saito, R., Kiyosawa, H., Kanai, A. and Tomita, M.

Gene　392 392 ( 1-2 ) 134-141 - 141 2007

Research paper (scientific journal), Joint Work, ISSN 03781119

Recent large-scale transcriptomic analyses have identified numerous endogenously encoded cis-antisense RNAs that are thought to play important roles in diverse cellular processes although comprehensive comparative studies among multiple species have yet to be performed. To investigate conserved genomic features across various species that may be related to sense-antisense regulation, we performed comparative analysis of approximately 1000-2000 cis-encoded antisense RNA pairs from five model eukaryotes (Homo sapiens, Mus musculus, Drosophila melanogaster, Arabidopsis thaliana, and Oryza sativa). Analysis of overlapping patterns relative to the exon-intron structure revealed that the number of pairs sharing the 3′ part of the transcripts was larger than that of the 5′-sharing pairs except in rice. Moreover, most of the well-conserved sense-antisense pairs between human and mouse exhibited 3′-overlaps, suggesting that regulatory mechanisms involving these regions may be important in sense-antisense transcription. Functional classification using Gene Ontology revealed that genes related to catalytic activity, nucleotide binding, DNA metabolism, and mitochondria were preferentially distributed within the set of exon-overlapping sense-antisense genes compared to the non-exon-overlapping group in animals. Despite the numerous sense-antisense pairs identified in human and mouse individually, the number of conserved pairs was extremely small (6.6% of the entire set). Whereas both genes of most of the conserved sense-antisense pairs had protein-coding potential, nearly half of the non-conserved pairs included a non-coding RNA, suggesting that non-coding sense-antisense RNAs may function in species-specific regulatory pathways. © 2006 Elsevier B.V. All rights reserved.

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The Hsp70 chaperone system maintains high concentrations of active proteins and suppresses ATP consumption during heat shock

Hu, B. and Tomita, M.

Systems and Synthetic Biology 1 ( 1 ) 47-58 - 58 2007

Research paper (scientific journal), Joint Work, ISSN 18725325

Hsp70 chaperones assist protein folding by cycling between the ATP-bound T state with low affinity for substrates and the ADP-bound R state with high affinity for substrates. The transition from the T to R state is catalyzed by the synergistic action of the substrate and DnaJ cochaperones. The reverse transition from the R state to the T state is accelerated by the nucleotide exchange factor GrpE. These two processes, T-to-R and R-to-T conversion, are affected differently by temperature change. Here we modeled Hsp70-mediated protein folding under permanent and transient heat shock based on published experimental data. Our simulation results were in agreement with in vitro wild-type Escherichia coli chaperone experimental data at 25°C and reflected R-to-T ratio dynamics in response to temperature effects. Our simulation results suggested that the chaperone system evolved naturally to maintain the concentration of active protein as high as possible during heat shock, even at the cost of recovered activity after return to optimal growth conditions. They also revealed that the chaperone system evolved to suppress ATP consumption at non-optimal high growing temperatures. © Springer Science + Business Media B.V. 2007.

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Prediction of liquid chromatographic retention times of peptides generated by protease digestion of the Escherichia coli proteome usina artificial neural networks

Shinoda K., Sugimoto M., Yachie N., Sugiyama N., Masuda T., Robert M., Soga T., Tomita M.

Journal of Proteome Research 5 ( 12 ) 3312 - 3317 2006.12

ISSN 15353893

We developed a computational method to predict the retention times of peptides in HPLC using artificial neural networks (ANN). We performed stepwise multiple linear regressions and selected for ANN input amino acids that significantly affected the LC retention time. Unlike conventional linear models, the trained ANN accurately predicted the retention time of peptides containing up to 50 amino acid residues. In 834 peptides, there was a strong correlation (R2= 0.928) between measured and predicted retention times. We demonstrated the utility of our method by the prediction of the retention time of 121 273 peptides resulting from LysC-digestion of the Escherichia coli proteome. Our approach is useful for the proteome-wide characterization of peptides and the identification of unknown peptide peaks obtained in proteome analysis. © 2006 American Chemical Society.

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Multiresolution analysis uncovers hidden conservation of properties in structurally and functionally similar protiens

Chua G.H., Krishnan A., Li K.B., Tomita M.

Journal of Bioinformatics and Computational Biology 4 ( 6 ) 1245 - 1267 2006.12

ISSN 02197200

Physicochemcial properties of amino acids are important factors in determining protein structure and function. Most approaches make use of averaged properties over entire domains or even proteins to analyze their structure or function. This level of coarseness tends to hide the richness of the variability in the different properties across functional domains. This paper studies the conservation of physicochemical properties in a functionally similar family of proteins using a novel wavelet-based technique known as multiresolution analysis. Such an analysis can help uncover characteristics that can otherwise remain hidden. We have studied the protein kinase family of sequences and our findings are as follows: (a) a number of different properties are conserved over the functional catalytic domain irrespective of the sequence identities; (b) conservation of properties can be observed at different frequency levels and they agree well with the known structural/ functional properties of the subdomains for the protein kinase family; (c) structural differences between the different kinase family members are reflected in the waveforms; and (d) functionally important mutations show distortions in the waveforms of conserved properties. The potential usefulness of the above findings in identifying functionally similar sequences in the twilight and midnight zones is demonstrated through a simple prediction model for the protein kinase family which achieved a recall of 93.7% and a precision of 96.75% in cross-validation tests. © Imperial College Press.

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HybGFS: a hybrid method for genome-fingerprint scanning

Shinoda, Kosaku ; Yachie, Nozomu ; Masuda, Takeshi ; Sugiyama, Naoyuki ; Sugimoto, Masahiro ; Soga, Tomoyoshi ; Tomita, Masaru

BMC Bioinformatics 7 8 2006.10

Research paper (scientific journal), Joint Work, ISSN 1471-2105

Background: Protein identification based on mass spectrometry (MS) has previously been performed using peptide mass fingerprinting (PMF) or tandem MS (MS/MS) database searching. However, these methods cannot identify proteins that are not already listed in existing databases. Moreover, the alternative approach of de novo sequencing requires costly equipment and the interpretation of complex MS/MS spectra. Thus, there is a need for novel high-throughput protein-identification methods that are independent of existing predefined protein databases. Results: Here, we present a hybrid method for genome-fingerprint scanning, known as HybGFS. This technique combines genome sequence-based peptide MS/MS ion searching with liquid-chromatography elution-time (LC-ET) prediction, to improve the reliability of identification. The hybrid method allows the simultaneous identification and mapping of proteins without a priori information about their coding sequences. The current study used standard LC-MS/MS data to query an in silico-generated six-reading-frame translation and the enzymatic digest of an entire genome. Used in conjunction with precursor/product ion-mass searching, the LC-ETs increased confidence in the peptide-identification process and reduced the number of false-positive matches. The power of this method was demonstrated using recombinant proteins from the Escherichia coli K12 strain. Conclusion: The novel hybrid method described in this study will be useful for the large-scale experimental confirmation of genome coding sequences, without the need for transcriptome-level expression analysis or costly MS database searching. © 2006 Shinoda et al; licensee BioMed Central Ltd.

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Evaluation of metabolic alteration in transgenic rice overexpressing dihydroflavonol-4-reductase

Takahashi, Hideyuki ; Hayashi, Mitsunori ; Goto, Fumiyuki ; Sato, Shigeru ; Soga, Tomoyoshi ; Nishioka, Takaaki ; Tomita, Masaru ; Kawai-Yamada, Maki ; Uchimiya, Hirofumi

Annals of Botany 98 ( 4 ) 819-825 - 825 2006.10

Research paper (scientific journal), Joint Work, ISSN 0305-7364

Background and Aims: Previous studies have shown that transgenic rice plants overexpressing YK1, which possesses dihydroflavonol-4-reductase (DFR) activity, showed biotic and abiotic stress tolerance. High throughput profiles of metabolites have also been shown in such transgenic plants by Fourier transform ion cyclotron mass spectrometry. In this study, capillary electrophoresis mass spectrometry analysis (CE/MS) was employed to identify precise metabolites such as organic acids, amino acids and sugars. Methods: Using CE/MS, we analysed several metabolites of glycolysis, the tricarboxylic acid (TCA) cycle and the pentose phosphate pathway. In addition, the concentrations of sugars and ion were quantified. Key Results: In YK1 (DFR)-overexpressing plants, the concentrations of cis-aconitate, isocitrate and 2-oxoglutarate were higher in leaves, whereas those of fructose-1,6- bisphosphate and glyceraldehyde-3-phosphate were lower in roots. In seeds, the amounts of free amino acids and metals were altered, whereas sugars in seeds were kept constant. In YK1 calli, an approx. 3-fold increase in glutathione was observed, whereas the activities of glutathione peroxidase and glutathione reductase were concomitantly increased. Conclusions: The overexpression of YK1 (DFR) was associated with slight changes in the amounts of several metabolites analysed in whole plants, whilst glutathione derivatives were substantially increased in suspension-cultured cells. © The Author 2006. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.

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