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The current research aims of our laboratory are to understand the mechanism and regulation of the production of useful metabolites in plants through the genomics and post-genomics approaches and subsequently to apply the obtained knowledge to genetic engineering in vivo.

Prof. Kazuki Saito

Faculty of Pharmaceutical Sciences

1. Construction of technology platforms of integration of transcriptomics and metabolomics, and its application to functional genomics in plants.

Metabolomics approach is applied to understand holistic picture of plant cells and identify the function of genes in plants. Technology platforms for integration of metabolomics by LC-MS, GC-MS, CE-MS, FT-MS and FT-NIR, and transcriptomics by DNA microarray is constructed through collaboration with several external groups. Since incorporation of bioinformatics is a key issue for data mining and simulation, efforts of collaboration are putting on this particular issue. Comprehensive metabolite isolation and identification from A. thaliana is also undertaken as a basis of metabolomics research. This platform of integrated metabolomics and transcriptomics is applying to decipher the function of unknown genes from a model plant Arabidopsis thaliana and useful medicinal plants.

2. Mechanism and regulation of sulfur metabolism in plants by post-genomics approach.

The genes encoding some enzymes in sulfur assimilation (cysteine synthesis) were isolated from plants and characterized at a molecular level. By using Arabidopsis thaliana as a model plant, we obtained the molecular information how the whole sulfur assimilation pathway is regulated through the post-genomics approach. In particular, we characterized the function of several different serine acetyltransferase, cysteine synthase, ATP sulfurylase and APS kinase from A. thaliana by in vitro study with recombinant proteins and by reverse genetics with analysis of T-DNA insertion mutants. Genetic modification of sulfur assimilation was achieved by using transgenic plants for the genes involved in the sulfur assimilation pathway. These studies will be applied to molecular engineering of metabolism of sulfur containing secondary plant products.

3. Molecular genetic characterization of chemotypes in medicinal plants: Flavonoid biosynthesis.

To elucidate the molecular basis of anthocyanin-producing and non-producing formas of a traditional medicinal plant Perilla frutescens, we have isolated a number of structural genes and regulatory genes for anthocyanin biosynthesis from P. frutescens. The genes for a glutathione S-transferase, a novel anthocyanin glucosyltransferase and mutant alleles of Myc transcriptional factor were isolated and characterized. We clarified the precise reaction mechanism of anthocyanidin synthase and related 2-oxoglutarate-dependent flavonoid oxygenases by combination of in vitro study by recombinant enzymes with 18O as a tracer and in silico calculation. The study of post-genome approach by transcriptomics and metabolomics with anthocyanin-overaccumulating A. thaliana mutant is also undertaken to identify the genes involved in anthocyanin production and storage and to reveal the molecular network.

4. Biochemistry and molecular biology of alkaloid biosynthesis in plants.

The enzymes involved in quinolizidine alkaloid formation in the Leguminosae plants were purified, and their enzymatic properties and distribution were investigated. We are currently studying to isolate cDNAs responsible for quinolizidine alkaloid biosynthesis from "bitter" and "sweet" varieties of Lupinus plants by differential screening between two varieties. The project for the production of anti-neoplastic camptothecin alkaloid in cell culture has been also conducted. We have established the hairy root culture that produced and excreted camptothecin, and isolated several genes involved in biosynthesis of camptothecin. We have isolated a set of genes which are specifically expressed only in the cells producing camptothecin and thus presumed to be involved in camptothecin formation by PCR-select subtraction. We are also investigating self-resistance mechanism of camptothecin-producing plants against toxicity of self-produced camptothecin.