Details of a Researcher - Tomita, Masaru

Papers - Tomita, Masaru

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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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The GC skew index: a measure of genomic compositional asymmetry and the degree of replicational selection

Arakawa, K. and Tomita, M.

Evolutionary Bioinformatics 3 159 - 168 2007

Research paper (scientific journal), Joint Work

Circular bacterial chromosomes have highly polarized nucleotide composition in the two replichores, and this genomic strand asymmetry can be visualized using GC skew graphs. Here we propose and discuss the GC skew index (GCSI) for the quantifi cation of genomic compositional skew, which combines a normalized measure of fast Fourier transform to capture the shape of the skew graph and Euclidean distance between the two vertices in a cumulative skew graph to represent the degree of skew. We calculated GCSI for all available bacterial genomes, and GCSI correlated well with the visibility of GC skew. This novel index is useful for estimating confi dence levels for the prediction of replication origin and terminus by methods based on GC skew and for measuring the strength of replicational selection in a genome.

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Robust effects of Tsr-CheBp and CheA-CheYp affinity in bacterial chemotaxis

Matsuzaki, Y., Kikuchi, S. and Tomita, M.

Artificial intelligence in medicine 41 ( 2 ) 145-150 - 150 2007

Research paper (scientific journal), Joint Work, ISSN 09333657

Objective: Cell motility and chemotaxis play a role in the virulence of pathogenic bacteria, such as escape from host immune responses. Escherichia coli chemotaxis provides a well-characterized model system for the bacterial chemotaxis network. Two features of E. coli chemotaxis include signal amplification and robustly accurate adaptation. Recent simulation studies with models considering the effects of other receptors have suggested possible mechanisms for signal amplification. Although precise adaptation to aspartate has been explained by conventional kinetic models, the adaptation behavior of models incorporating the effects of other receptors remains unclear. Methods: We concentrated on how receptor crosstalk affects minimization of adaptation error and compared models in which the contribution of other receptors varied. Results: We demonstrated that the model is adaptable to attractant concentrations ranging from 0.1μ M to 10 mM with a decreased error rate (from 8% to 2%) when the kinetic constant of CheA and phosphorylated CheY dissociation is increased. Conclusion: The results suggest that accurate adaptation is maintained through control of both the interaction of cytoplasmic Che proteins and the activity of the receptor complex. © 2007 Elsevier B.V. All rights reserved.

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Microelectrospray interface with coaxial sheath flow for high-resolution capillary electrophoresis/mass spectrometry separation

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

Rapid Communications in Mass Spectrometry 21 ( 22 ) 3579 - 3584 - 3584 2007

Research paper (scientific journal), Joint Work, ISSN 09514198

We have fabricated a coaxial sheath liquid flow microelectrospray ionization (μESI) interface for capillary electrophoresis coupled with mass spectrometry (CE/MS). The ESI interface, which features a reduced probe diameter (130 μm i.d. x 174 μm o.d.) with a nebulizer-free format, can relatively easily electrospray a large amount of make-up sheath liquid (5-10 μL/min) over the long term (more than 80 runs) with a high degree of stability. The interface also provides higher separation qualities and improved detection sensitivities compared with a conventional ion spray (IS) interface. Copyright © 2007 John Wiley & Sons, Ltd.

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Permuted tRNA Genes Expressed via a Circular RNA Intermediate in Cyanidioschyzon merolae

Soma, A., Onodera, A., Sugahara, J., Kanai, A., Yachie, N., Tomita, M., Kawamura, F., Sekine, Y.

Science 318 ( 5849 ) 450-453 - 453 2007

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

A computational analysis of the nuclear genome of a red alga, Cyanidioschyzon merolae, identified 11 transfer RNA (tRNA) genes in which the 3′ half of the tRNA lies upstream of the 5′ half in the genome. We verified that these genes are expressed and produce mature tRNAs that are aminoacylated. Analysis of tRNA-processing intermediates for these genes indicates an unusual processing pathway in which the termini of the tRNA precursor are ligated, resulting in formation of a characteristic circular RNA intermediate that is then processed at the acceptor stem to generate the correct termini.

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Model based definition of population heterogeneity and its effects on metabolism in sporulating Bacillus subtilis

Morohashi, M., Ohashi, Y., Tani, S., Ishii, K., Itaya, M., Nanamiya, H., Kawamura, F., Tomita, M. and Soga, T.

Journal of Biochemistry 142 ( 2 ) 183-191 - 191 2007

Research paper (scientific journal), Joint Work, ISSN 0021924X

The soil bacterium Bacillus subtilis forms dormant, robust spores as a tactic to ensure survival under conditions of starvation. However, the sporulating culture includes sporulating and non-sporulating cells, because a portion of the cell population initiates sporulation in wild-type strain. We anticipated that the population effect must be considered carefully to analyse samples yielding population heterogeneity. We first built a mathematical model and simulated for signal transduction of the sporulation cue to see what mechanisms are responsible for generating the heterogeneity. The simulated results were confirmed experimentally, where heterogeneity is primarily modulated by negative feedback circuits, resulting in generation of a bistable response within the sporulating culture. We also confirmed that mutants relevant to negative feedback yield either sporulating or non-sporulating subpopulations. To see the effect of molecular mechanism between sporulating and non-sporulating cells in distinct manner, metabolome analysis was conducted using the above mutants. The metabolic profiles exhibited distinct characteristics with time regardless of whether sporulation was initiated or not. In addition, several distinct characteristics of metabolites were observed between strains, which was inconsistent with previously reported data. The results imply that careful consideration must be made in the interpretation of data obtained from cells yielding population heterogeneity. © 2007 The Japanese Biochemical Society.

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Variation in the Correlation of G + C Composition with Synonymous Codon Usage Bias among Bacteria

Suzuki H, Saito R, Tomita M.

EURASIP J Bioinform Syst Biol. 2007 ( 61374 ) 2007

Research paper (scientific journal), Joint Work, ISSN 16874145

G + C composition at the third codon position (GC3) is widely reported to be correlated with synonymous codon usage bias. However, no quantitative attempt has been made to compare the extent of this correlation among different genomes. Here, we applied Shannon entropy from information theory to measure the degree of GC3 bias and that of synonymous codon usage bias of each gene. The strength of the correlation of GC3 with synonymous codon usage bias, quantified by a correlation coefficient, varied widely among bacterial genomes, ranging from -0.07 to 0.95. Previous analyses suggesting that the relationship between GC3 and synonymous codon usage bias is independent of species are thus inconsistent with the more detailed analyses obtained here for individual species.

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Proteome-Wide Prediction of Novel DNA/RNA-Binding Proteins Using Amino Acid Composition and Periodicity in the Hyperthermophilic Archaeon Pyrococcus furiosus

Fujishima, K., Komasa, M., Kitamura, S., Suzuki, H., Tomita, M. and Kanai, A.

DNA Research 14 ( 3 ) 91-102 - 102 2007

Research paper (scientific journal), Joint Work, ISSN 13402838

Proteins play a critical role in complex biological systems, yet about half of the proteins in publicly available databases are annotated as functionally unknown. Proteome-wide functional classification using bioinformatics approaches thus is becoming an important method for revealing unknown protein functions. Using the hyperthermophilic archaeon Pyrococcus furiosus as a model species, we used the support vector machine (SVM) method to discriminate DNA/RNA-binding proteins from proteins with other functions, using amino acid composition and periodicities as feature vectors. We defined this value as the composition score (CO) and periodicity score (PD). The P. furiosus proteins were classified into three classes (I-III) on the basis of the two-dimensional correlation analysis of CO score and PD score. As a result, approximately 87 of the functionally known proteins categorized as class I proteins (CO score + PD score > 0.6) were found to be DNA/RNA-binding proteins. Applying the two-dimensional correlation analysis to the 994 hypothetical proteins in P. furiosus, a total of 151 proteins were predicted to be novel DNA/RNA-binding protein candidates. DNA/RNA-binding activities of randomly chosen hypothetical proteins were experimentally verified. Six out of seven candidate proteins in class I possessed DNA/RNA-binding activities, supporting the efficacy of our method. © The Author 2007. Kazusa DNA Research Institute.

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Direct measurement of isotopomer of intracellular metabolites using capillary electrophoresis time-of-flight mass spectrometry for efficient metabolic flux analysis

Toya, Y., Ishii, N., Hirasawa, T., Tomita, M. and Soga, T.

Journal of Chromatography A 1159 ( 1-2 ) 134–141 - 141 2007

Research paper (scientific journal), Joint Work, ISSN 00219673

We have developed a metabolic flux analysis method that is based on 13C-labeling patterns of the intracellular metabolites directly measured by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). The flux distribution of the central carbon metabolism in Escherichia coli was determined by this new approach and the results were compared with findings obtained by conventional GC-MS analysis based on isotopomer of the proteinogenic amino acids. There were some differences in estimation results between new approach using CE-TOFMS and conventional approach using GC-MS. These were thought to be attributable to variations in measured mass distributions between amino acids and the corresponding precursors and to differences in the sensitivity of the exchange coefficients to mass distributions. However, our CE-TOFMS method facilitates high-throughput flux analysis without requiring complicated sample preparation such as hydrolysis of proteins and derivatization of amino acids. © 2007 Elsevier B.V. All rights reserved.

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Analysis of nucleotides by pressure-assisted capillary electrophoresis　mass spectrometry using silanol mask technique

Soga, T., Ishikawa, T., Igarashi, S., Sugawara, K., Kakazu, Y. and Tomita, M.

Journal of Chromatography A 1159 ( 1-2 ) 125–133 - 133 2007

Research paper (scientific journal), Joint Work, ISSN 00219673

A method for the determination of nucleotides based on pressure-assisted capillary electrophoresis-electrospray ionization mass spectrometry (PACE-MS) is described. To prevent multi-phosphorylated species from adsorbing onto the fused-silica capillary, silanol groups were masked with phosphate ions by preconditioning the capillary with the background electrolyte containing phosphate. During preconditioning, nebulizer gas was turned off to avoid contamination of MS detector with phosphate ions. To detect nucleotides using the CE positive mode at a pH 7.5, it was necessary to apply air pressure to the inlet capillary during electrophoresis to supplement the electroosmotic flow (EOF) toward the cathode. Moreover, we exchanged the running electrolyte every analysis using the buffer replenishment system to obtain the required reproducibility. Under the optimized conditions, 14 phosphorylated species such as nucleotides, nicotinamide-adenine dinucleotides and coenzyme A (CoA) compounds were well determined in less than 20 min. The relative standard deviations (n = 6) of the method were better than 0.9% for migration times and between 1.7% and 8.1% for peak areas. The detection limits for these species were between 0.5 and 1.7 μmol/L with pressure injection of 50 mbar for 30 s (30 nL) at a signal-to-noise ratio of 3. This approach is robust and quantitative compared to the previous method, and its utility is demonstrated by the analysis of intracellular nucleotides and CoA compounds extracted from Escherichia coli wild type, pfkA and pfkB knockout mutants. The methodology was used to suggest that pfkA is the main functional enzyme. © 2007 Elsevier B.V. All rights reserved.

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Conjugational transfer system to shuttle giant DNA cloned by Bacillus subtilis genome (BGM) vector

Kuroki, A., Ohtani, N., Tsuge, K., Tomita, M. and Itaya, M.

Gene 399 ( 1 ) 72-80 - 80 2007

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

The Bacillus subtilis GenoMe (BGM) vector was designed as a versatile vector for the cloning of giant DNA segments. Cloned DNA in the BGM can be retrieved to a plasmid using our Bacillus recombinational transfer (BReT) method that takes advantage of competent cell transformation. However, delivery of the plasmid to a different B. subtilis strain by the normal transformation method is hampered by DNA size-related inefficiency. Therefore, we designed a novel method, conjugational plasmid-mediated DNA retrieval and transfer (CReT) from the BGM vector, and investigated conjugational transmission to traverse DNA between cells to circumvent the transformation-induced size limitation. pLS20, a 65-kb plasmid capable of conjugational transfer between B. subtilis strains, was modified to retrieve DNA cloned in the BGM vector by homologous recombination during normal culture. As the plasmid copy number was estimated to be 3, the retrieval plasmid was selected using increased numbers of marker genes derived from the retrieved DNA. We applied this method to retrieve Synechocystis genome segments up to 90 kb in length. We observed retrieved plasmid transfers between B. subtilis strains by conjugation in the absence of structural alterations in the DNA fragment. Our observations extend DNA transfer protocols over previously exploited size ranges. © 2007 Elsevier B.V. All rights reserved.

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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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