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.

Full-length cDNAs play an essential role in identifying genes and determining their promoter regions. Here we describe a simple method for constructing a full-length cDNA library, which has the following advantages: (i) it consists of only three steps including direct ligation between a vector and a cDNA strand using T4 RNA ligase, (ii) it contains neither a PCR process generating mutations nor restriction enzyme treatment causing truncation of cDNA, (iii) the intactness of cDNA is assured due to the presence of an additional dGMP at its 5' end, (iv) approximately 95% of cDNA clones are full-length when cultured cells or fresh tissues are used, (v) several micrograms of total RNA without mRNA purification is sufficient for preparation of a library containing >10(5) independent clones, and (vi) a long-sized full-length cDNA up to 9.5 kbp can be cloned. This method will accelerate comprehensive gene analysis in a variety of eukaryotes.