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Results and Future Plans

Former Plant Functional Genomics Research Group
Collection and Analysis of <em>Arabidopsis thaliana</em> mutants to Identify Genes Useful for Improving Productivity

Kazuo Shinozaki

Former Project Director of the Plant Functional Genomics Research Group and current Director of the RIKEN Plant Science Center

Professor Shinozaki was the former Project Director of the Plant Functional Genomics Research Group, directing research on the plant genome (from 1999). He is currently Director of the RIKEN Plant Science Center in Yokohama, and is responsible for promoting research on development of improved plant productivity related to sustainable food supply, energy production, and environmental conservation.

Arabidopsis thaliana can be grown easily in the laboratory environment and its seeds require about 3 months to be ready for harvest. It has been widely used as a model organism in studies of genes related to the physiological functions of plants. In addition, A. thaliana possesses the smallest genome among all higher plants. In the 1990s, genomics studies on A. thaliana as a plant model were developed worldwide, and in 2000, concerted international efforts (including contributions by Japan) led to the complete sequencing of the A.thaliana genome.
Based on the genome sequencing data, it was predicted that the A. thaliana genome contains about 27,000 protein-encoding genes. However, the functions and expression of most of these genes remain unclear. To analyze their functions, it was important to collect the gene knock-out mutants and the full-length cDNAs for all genes, and then to develop the comprehensive analysis systems using the genome resources.
In 1999, Dr. Minani Matsui (current Group Director of the Plant Functional Genomics Research Group, RIKEN Plant Science Center) joined the former Plant Functional Genomics Research Group based at GSC. In 2006, the group was re-located to its current site at the Plant Science Center.

Diverse methods for generating mutant strains

In the first 5 years, we created a pool of 18,000 mutants with genetic alterations via transposon insertion and constructed a database detailing the flanking sequences. For gain-of-function mutants with single additional heterologous functions, we generated mutants inserted with a transcription enhancer sequence and created activation tagging lines*1 for over 70,000 mutant strains. Based on such groundwork, our group has carried out studies on diverse topics including environmental response, chloroplast functions, and morphogenesis, while we have deposited our resources to the RIKEN BioResource Center, for providing support to researchers here in Japan and overseas.
Transposon-tagged mutants have basically single insertion in the genome, which is an advantage to investigate gene functions based on mutant phenotypes. Now we are proceeding to collect "single-gene mutant lines" for every protein-coding gene of Arabidopsis. A comprehensive phenotype analysis to reveal gene function, i, e, "phenome analysis", is still in progress at RIKEN Plant Science Center.

*1 Activation tagging
Mutant strains are created by inserting a T-DNA (tag) containing an enhancer into the plant genomic sequence. The T-DNA is randomly integrated into the host plant's genome, using Agrobacterium tumefaciens as a carrier to infect the plant. The heterologous enhancer sequence induces overexpression of protein products of genes near the site of T-DNA insertion, indicating that the gene mutation results in a dominant phenotype. In addition, by identifying the genes adjacent to the inserted T-DNA, the causative genes for phenotypic alterations can be specifically determined.

Various mutant strains in the A. thaliana collection

A large variety of mutant strains have been created using methods such as activation tagging and transposon insertion. So far, the collection includes about 100,000 types of mutant strains, which exhibit phenotypic variations such as different leaf shape and size or delayed flowering.

Large-scale collection of 18,000 full-length cDNAs

Genome sequencing projects predicted the presence of the genes, however, it was difficult to predict the expressed genes faithfully from the genome sequence data. So we tried to isolate the A. thaliana full-length cDNAs to identify the structure of the expressed genes. The full-length cDNA is also an important tool for the functional analysis of the protein.
We have collected about 18,000 RIKEN Arabidopsis full-length cDNA (RAFL cDNA) groups (about 60% of the predicted genes) and carried out functional annotation of them in collaboration with the Genome Exploration Research Group. This is the largest collection of A. thaliana full-length cDNAs. Additionally, to maximize the utility of the RAFL cDNA collection for domestic and overseas researchers, we have distributed the full-length cDNA and mutant resources to the research community in collaboration with the Experimental Plant Division of RIKEN BioResource Center (BRC). Consequently, our genomic resources have been made globally available, contributing directly to the reputation of RIKEN at the international level.
At the same time, in order to improve efficiency in the use of full-length cDNA for genetics analyses, we have produced transgenic plants with additional heterologous functions and carried out screening for multiple mutant strains. For example, we developed a novel screening system of gene functions, the FOX (Full-length cDNA OvereXpressor gene) hunting system, which makes use of full-length cDNA overexpressed in the plants. Using this technology, we can perform analysis on the causative genes responsible for phenotypic alterations seen in transgenic plants.

FOX hunting system

This system efficiently generates mutant plant strains of diverse phenotypes with enhanced expression of specific genes of interest. It uses an "Agrobacterium-based library" comprising full-length plant cDNAs for one-time genetic transformation. Full-length cDNA available for study can be used to rapidly determine the effect of hyper- or hypoexpression of a gene. In this way, causative genes for phenotype alterations can be identified without the need for analysis of the genomic sequence.

The FOX hunting system as a research tool is unique among RIKEN's mutant strain resources, in that it exploits the advantages of integrating the use of full-length cDNA and generation of transgenic plants. In addition, we have teamed up with the National Institute of Agrobiological Sciences (under the Ministry of Agriculture, Forestry and Fisheries) to create plant lines with full-length rice cDNA via the same system, allowing us to successfully conduct functional studies on rice genes overexpressed in A. thaliana.

Toward integrative understanding and applications of plant life

With progress in decoding the genome sequences of various plants, we have entered a new phase of plant research based on accumulated genomics findings. In plant genomics studies, the topics deserving high priorities are "elucidation of gene functions of the plant genome", "gene expression profile analysis", "gene expression network analysis for high-order gene interactions", and "protein network analysis for protein-protein interactions between gene products". In the plant world, it has been suggested that there exist a rich variety of metabolites (about 200,000 types in all plants, and 5000 in A. thaliana). In this area, we have been engaged in comprehensive profiling analysis of sugars, lipids, amino acids, and secondary metabolites. In order to achieve greater understanding of life in nature, it is necessary to conduct large-scale studies covering the areas of genomics studies, gene expression analyses (in the form of transcriptomics studies), protein profiling analyses (in the form of proteomics studies), and small-molecule metabolite profiling analyses (in the form of metabolomics studies), which collectively form the focus of research being undertaken at the RIKEN Plant Science Center.
In the future, using the foundation laid for basic research on A. thaliana, we hope to expand the scope of our studies to include applied research using the full-length cDNAs from the crops and trees. We have also drawn up a 15-year plan for sustainable environment to guide future research projects seeking to provide solutions to problems with global impact, including those concerning food supply, energy, biomass production, and the environment.

Illustration of the knockout-type phenotype database

A public access database has been made containing 4000 systematically classified mutant strains, information on causative genes and illustrative images. Users can find many phenotype anomalies related to seed size, flowering, or other newly identified mutants that are agriculturally important.