Since the completion of genome sequences of model organisms, functional identification of unknown genes has become a principal challenge in biology. Postgenomics sciences such as transcriptomics, proteomics, and metabolomics are expected to discover gene functions. This report outlines the elucidation of gene-togene and metabolite-to-gene networks via integration of metabolomics with transcriptomics and presents a strategy for the identification of novel gene functions. Metabolomics and transcriptomics data of Arabidopsis grown under sulfur deficiency were combined and analyzed by batch-learning self-organizing mapping. A group of metabolites/genes regulated by the same mechanism clustered together. The metabolism of glucosinolates was shown to be coordinately regulated. Three uncharacterized putative sulfotransferase genes clustering together with known glucosinolate biosynthesis genes were candidates for involvement in biosynthesis. In vitro enzymatic assays of the recombinant gene products confirmed their functions as desulfoglucosinolate sulfotransferases. Several genes involved in sulfur assimilation clustered with O-acetylserine, which is considered a positive regulator of these genes. The genes involved in anthocyanin biosynthesis clustered with the gene encoding a transcriptional factor that up-regulates specifically anthocyanin biosynthesis genes. These results suggested that regulatory metabolites and transcriptional factor genes can be identified by this approach, based on the assumption that they cluster with the downstream genes they regulate. This strategy is applicable not only to plant but also to other organisms for functional elucidation of unknown genes.In the era of post-genomics, a systematic and comprehensive understanding of the complex events of life is a great concern in biology. The first step in this process is to identify all gene functions and gene-to-gene networks as the components of the system, the whole events of life. The metabolome is the final product of a series of gene actions. Hence, metabolomics has a potential to elucidate gene functions and networks, especially when integrated with transcriptomics. A promising approach is pair-wise transcript-metabolite correlation analysis, which can reveal unexpected correlations and bring to light candidate genes for modifying the metabolite content (1). Gene functions involved in the specific pathway of interest have been identified by the integration of transcript and targeted metabolic profiling in experimental systems where the pathway was activated (2-6). However, up to now, no gene function has been identified by non-targeted analyses of the transcriptome and metabolome. In this report, we analyzed the non-targeted metabolome and transcriptome of a model plant Arabidopsis under sulfur (S) 1 deficiency whose genome sequencing has been completed. Our strategy for integrated analyses using batch-learning-selforganizing mapping (BL-SOM) (7-9) enabled the identification of gene-to-gene and metabolite-to-gene networks and new gene fun...

All members of the sulphotransferase (SOT, EC 2.8.2.-) protein family use 3'-phosphoadenosine 5'-phosphosulphate (PAPS) as the sulphuryl donor and transfer the sulphonate group to an appropriate hydroxyl group of several classes of substrates. These enzymes have highly conserved domains and can be found in eubacteria and eukaryotes. In mammals, sulphate conjugation catalysed by SOTs constitutes an important reaction in the transformation of xenobiotics, and in the modulation of the biological activity of steroid hormones and neurotransmitters. In plants, sulphate-conjugation reactions seem to play an important role in plant growth, development, and adaptation to stress. To date only a few plant SOTs have been characterized in detail. The flavonol 3- and 4'-SOTs from Flaveria species (Asteraceae), which catalyse the sulphonation of flavonol aglycones and flavonol 3-sulphates, respectively, were the first plant SOTs for which cDNA clones were isolated. The plasma membrane associated gallic acid SOT of Mimosa pudica L. pulvini cells may be intrinsic to signalling events that modify the seismonastic response. In Brassica napus L. a SOT catalyses the O-sulphonation of brassinosteroids and thereby abolishes specifically the biological activity of 24-epibrassinolide. The fully sequenced genome of Arabidopsis thaliana Heynh. contains in total 18 genes that are likely to encode SOT proteins based on sequence similarities of the translated products with an average identity of 51.1%. So far only one SOT from A. thaliana (At5g07000) was functionally characterized: the protein was shown to catalyse the sulphonation of 12-hydroxyjasmonate and thereby inactivate excess jasmonic acid in plants. The substrates and, therefore, the physiological roles of SOTs are very diverse. By using the numerous informative databases and methods available for the model plant A. thaliana, the elucidation of the functional role of the SOT protein family will be accelerated.

Glucosinolates are a group of over 130 nitrogencontaining and sulfur-containing natural products found in vegetative and reproductive tissues of 16 plant families, but are most well known as major secondary metabolites in the Brassicaceae [1,2]. In Arabidopsis thaliana (L.) Heynh., nearly 30 different glucosinolates have been described [1,3,4] Sulfotransferases (SOTs) catalyse the transfer of a sulfate group from 3¢-phosphoadenosine 5¢-phosphosulfate (PAPS) to an appropriate hydroxy group of various substrates with the parallel formation of 3¢-phosphoadenosine 5¢-phosphate. In Arabidopsis thaliana, 18 SOT proteins (AtSOT) have been identified. Three of them, AtSOT16, AtSOT17 and AtSOT18, catalyse the sulfation of desulfoglucosinolates. The proteins were expressed in Escherichia coli, purified by affinity chromatography and used for enzyme kinetic studies. By establishing two types of enzyme assay using both 35 S-labelled and unlabelled PAPS, separation of the products by HPLC, and detection of the products by monitoring radioactivity or UV absorption, the substrate specificities of the three AtSOT proteins were determined. They show different maximum velocities with several desulfoglucosinolates as substrates and differ in their affinity for desulfobenzylglucosinolate and PAPS. The sequences encoding AtSOT18 were amplified from Arabidopsis ecotypes C24 and Col0; the two expressed proteins differ in two out of 350 amino acids. These amino-acid variations led to different substrate specificities. Exchange of one of the two amino acids in AtSOT18 from C24 to the respective amino acid in AtSOT18 from Col0 gave the C24 protein the same substrate specificity as the wild-type AtSOT18 protein from Col0. All three desulfoglucosinolate AtSOT proteins are localized in the cytoplasm, as demonstrated by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts. Northern blot analysis indicated differential expression of the three AtSOT genes in plant organs and tissues at different developmental stages and during a light ⁄ darkness cycle. High (500 lm) and low (50 lm) sulfate concentrations in the medium did not influence the levels of expression.Abbreviations ER, endoplasmic reticulum; GFP, green fluorescent protein; PAP, phosphoadenosine 5¢-phosphate; PAPS, 3¢-phosphoadenosine 5¢-phosphosulfate; SOT, sulfotransferase.

Sulfotransferases (SOTs) (EC 2.8.2.-) catalyze the transfer of a sulfate group from the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to a hydroxyl group of different substrates. In Arabidopsis thaliana, three SOTs were identified to catalyze the last step of glucosinolate (Gl) core structure biosynthesis called AtSOT16, 17 and 18. These enzymes from Arabidopsis ecotype C24 were overexpressed in Escherichia coli and purified by affinity chromatography. Recombinant proteins were used to determine substrate specificities to investigate whether each of the three desulfo (ds)-Gl SOTs might influence the Gl pattern of Arabidopsis differently. After optimization of the enzyme assay, it was possible to measure in vivo substrates with non-radioactive PAPS by HPLC analysis of the product. In vitro enzyme assays revealed a preference of AtSOT16 for the indolic ds-Gl indol-3-yl-methyl, AtSOT17 showed an increased specific activity with increasing chain length of ds-Gl derived from methionine and AtSOT18 preferred the long-chain ds-Gl, 7-methylthioheptyl and 8-methylthiooctyl, derived from methionine. In planta ds-Gl exist side by side; therefore, initial results from one substrate measurements were verified using a defined mixture of ds-Gl and ds-Gl/Gl leaf extracts from Arabidopsis ecotype C24. These studies confirmed the one substrate measurements. To compare SOTs from different Arabidopsis ecotypes, additionally, AtSOT18* from ecotype Col-0 was overexpressed in E. coli and purified. The recombinant protein was used for in vitro measurements and revealed a different enzymatical behavior compared with AtSOT18 from C24. In conclusion, there are differences in the substrate specificities between the three ds-Gl AtSOT proteins within ecotype C24 and differences among ds-Gl AtSOT18 proteins from different ecotypes.

A coliphage M13 chimer containing the Saccharomyces cerevisiae TRPI gene and ARSJ replication origin (mPY2) was grown on an ung-dut-strain of Escherichia coli. The resulting single-stranded phage DNA had 13% of thymine residues substituted by uracil. This DNA failed to transform a Atrpl yeast strain to prototrophy. However, when a mutagenized yeast stock was transformed with uracil-containing singlestranded mPY2 DNA, unstable transformants were obtained. After plasmid segregation, about half of these were retransformed at a high frequency by uracil-containing single-stranded mPY2 DNA. In vitro, these mutants were defective for uracil-DNA-glycosylase activity. They were designated ungi. Strains containing the ungi mutation have an increased sensitivity to sodium bisulfite and sodium nitrite but a wild-type sensitivity to methyl methanesulfonate, UV light, and drugs that cause depletion of the thymidylate pool. They have a moderate mutator phenotype for nuclear but not for mitochondrial genes. A low mitochondrial uracil-DNAglycosylase activity was demonstrated in the mutant strains.Uracil is introduced into DNA either during DNA replication by incorporation of dUMP instead of dTMP or by spontaneous or induced deamination of cytosine to uracil (10,19,28,30,33,39). From both of these modifications, although different in nature (i.e., a dA-dU base pair as a result of misincorporation versus a dG. dU base pair through deamination) uracil is efficiently excised by the enzyme uracil-DNA-glycosylase. This enzyme has been isolated from a wide variety of procaryotic and eucaryotic organisms (16). It is typically a monomeric enzyme with a molecular weight of 20,000 to 30,000, equally active on single-stranded (ss) and double-stranded (ds) DNAs (16). Unlike nucleases, which require Mg2+ for activity, uracil-DNA-glycosylase is fully active in the presence of EDTA. This facilitates activity measurements in crude extracts containing nucleases.Escherichia coli mutants (ung) deficient for uracil-DNAglycosylase have been useful in the study of the mechanism of DNA replication (24, 39) and in delineating excision repair pathways (36). Similarly, such a mutant in the yeast Saccharomyces cerevisiae could help in the study of analogous problems. To our knowledge all uracil-DNA-glycosylase mutants have been obtained by enzymatic screening of extracts from large numbers of colonies after mutagenesis. This approach, however, tends to generate predominantly leaky mutants, as indeed has been observed for E. coli, Bacillus subtilis, and the smut fungus Ustilago maydis (13,29,42). In addition, in vivo enzymatic activities may be different from those measured in vitro.The observation by Duncan et al. (13), however, that uracil-containing T4 phage will plate efficiently only on E. coli ung mutants with an in vitro enzymatic activity of <1% of that of the wild type led us to devise a genetic scheme to isolate uracil-DNA-glycosylase mutants in S. cerevisiae. Using a genetic transformation procedure, we selected for survival of the transforming D...