SummaryThe integration of metabolomics and transcriptomics can provide precise information on gene-to-metabolite networks for identifying the function of unknown genes unless there has been a post-transcriptional modification. Here, we report a comprehensive analysis of the metabolome and transcriptome of Arabidopsis thaliana over-expressing the PAP1 gene encoding an MYB transcription factor, for the identification of novel gene functions involved in flavonoid biosynthesis. For metabolome analysis, we performed flavonoid-targeted analysis by high-performance liquid chromatography-mass spectrometry and non-targeted analysis by Fourier-transform ion-cyclotron mass spectrometry with an ultrahigh-resolution capacity. This combined analysis revealed the specific accumulation of cyanidin and quercetin derivatives, and identified eight novel anthocyanins from an array of putative 1800 metabolites in PAP1 over-expressing plants. The transcriptome analysis of 22 810 genes on a DNA microarray revealed the induction of 38 genes by ectopic PAP1 overexpression. In addition to well-known genes involved in anthocyanin production, several genes with unidentified functions or annotated with putative functions, encoding putative glycosyltransferase, acyltransferase, glutathione S-transferase, sugar transporters and transcription factors, were induced by PAP1. Two putative glycosyltransferase genes (At5g17050 and At4g14090) induced by PAP1 expression were confirmed to encode flavonoid 3-O-glucosyltransferase and anthocyanin 5-O-glucosyltransferase, respectively, from the enzymatic activity of their recombinant proteins in vitro and results of the analysis of anthocyanins in the respective T-DNA-inserted mutants. The functional genomics approach through the integration of metabolomics and transcriptomics presented here provides an innovative means of identifying novel gene functions involved in plant metabolism.

Plant metabolism is a complex set of processes that produce a wide diversity of foods, woods, and medicines. With the genome sequences of Arabidopsis and rice in hands, postgenomics studies integrating all ''omics'' sciences can depict precise pictures of a whole-cellular process. Here, we present, to our knowledge, the first report of investigation for gene-to-metabolite networks regulating sulfur and nitrogen nutrition and secondary metabolism in Arabidopsis, with integration of metabolomics and transcriptomics. Transcriptome and metabolome analyses were carried out, respectively, with DNA macroarray and several chemical analytical methods, including ultra high-resolution Fourier transform-ion cyclotron MS. Mathematical analyses, including principal component analysis and batch-learning self-organizing map analysis of transcriptome and metabolome data suggested the presence of general responses to sulfur and nitrogen deficiencies. In addition, specific responses to either sulfur or nitrogen deficiency were observed in several metabolic pathways: in particular, the genes and metabolites involved in glucosinolate metabolism were shown to be coordinately modulated. Understanding such geneto-metabolite networks in primary and secondary metabolism through integration of transcriptomics and metabolomics can lead to identification of gene function and subsequent improvement of production of useful compounds in plants. P lants produce a huge array of compounds used for foods, medicines, flavors, and industrial materials. These plant metabolites are synthesized and accumulated by the networks of proteins encoded in the genome of each plant. However, even after the completion of the genome sequencing of Arabidopsis (1) and rice (2, 3), function of those genes and networks of gene-to-metabolite are largely unknown. To reveal the function of genes involved in metabolic processes and gene-to-metabolite networks, the metabolomics-based approach is regarded as a direct way (4-7). In particular, integration of comprehensive gene expression profile with targeted metabolite analysis is shown to be an innovative way for identification of gene function for specific product accumulation in plant (8) and microorganisms (9). However, to depict a whole-cellular process of metabolism, integration of comprehensive gene expression analysis (transcriptomics), and nontargeted metabolite profiling (metabolomics) is needed. Bioinformatics designed suitably for data mining helps the integration efficiently.The gene expression profiling can be achieved by DNA array analysis. For metabolomics, a nontargeted, high-throughput analytical system is required. Traditionally, GC-MS has been used to detect Ͼ300 metabolites in plant tissues (5, 6). Fourier transform-ion cyclotron MS (FT-MS) is a system for metabolome analysis in which crude plant extract is introduced by means of direct injection without prior separation of metabolites by chromatography (10). The mass resolution (Ͼ100,000) and accuracy (Ͻ1 ppm) of FT-MS is extremely high; hence, comple...

SummaryTo investigate the changes in profiles of mRNA accumulation in response to sulfur deficiency, approximately 13 000 non-redundant Arabidopsis thaliana ESTs corresponding to approximately 9000 genes were analyzed using DNA macroarray. Three-week-old Arabidopsis plants grown on an agarose-solidified control medium were transferred to a sulfate-free medium and grown for 48 h for the analyses of sulfur-related metabolites and global gene expression profiles. Concentrations of sulfate, O-acetyl-L-serine (OAS), a positive regulator of sulfur deficiency-responsive genes, cysteine and glutathione (GSH) were determined. Plants transferred to sulfate-free media had reduced concentrations of sulfate and GSH, and OAS concentrations increased. Macroarray analysis revealed a number of genes, including APR2 and Sultr1;2, whose mRNA accumulation was increased by sulfur deficiency. Profiling was also carried out with plants treated with OAS under sulfate-sufficient condition. Scatter plot analysis revealed a positive correlation between the changes of expression levels by sulfur deficiency and by OAS treatment among the clones tested, suggesting that mRNA accumulation of a number of genes under sulfur deficiency is mainly controlled by OAS concentrations in tissues. It was also revealed that the sets of genes regulated under sulfur deficiency in leaves and roots differ considerably.