Studying the metabolome to produce plants useful to mankind
30 November 2007 (Volume 2 Issue 11)
One of the main targets of the second stage project of the RIKEN Plant Science Center (PSC) is to elucidate all the possible various metabolites produced by plants, and to associate them with genes. The Metabolomics Research Group led by Kazuki Saito, Group Director, is playing a key role in the project. The word ‘metabolome’ means the collection of all the small metabolites. It is expected that metabolomics research will enable the production of vegetables that contain a large amount of cancer-preventive agents and new functional metabolites useful for maintaining good health.
Pioneer in metabolomics research
These days, we often overhear the words, ‘metabolic disorder syndrome’. However, what do the words ‘metabolome’, or ‘metabolomics’ as in the ‘Metabolomics Research Group’ mean? “These new words were coined at the end of the 1990s,” explains Saito. ‘Metabolomics’ is the study of all metabolites, and the metabolome is the collection of all the metabolites. The word ‘genome’ refers to all the genetic information in an organism, ‘transcriptome’ to all substances transcribed from the DNA, ‘proteome’ to all the proteins, and ‘metabolome’ to all the metabolites produced in the body by proteins such as enzymes. These four areas are comprehensively referred to as ‘ome-science’, where ‘ome’ is a Latin word that means ‘a whole set’.
‘Metabolome’ is the most important keyword in the second stage project of the RIKEN PSC, which began in 2005. Why is the metabolome so important for the future of plant science?
Plants use photosynthesis to produce various metabolites including carbohydrates, such as starch and cellulose, amino acids, lipids, and secondary metabolites such as flavonoids and alkaloids. We utilized these metabolites as food, industrial and energy-source materials, drugs and medicines, and health functional food. Since plants cannot move, they produce various metabolites to cope with foreign enemies and environmental changes.
Plants have a variety of applications because they can produce various metabolites—substances that support their survival strategies. “To understand and utilize plants, it will become very important to understand the metabolism of plants,” says Saito.
Saito continues, “I think we were the first in Japan to start a project that highlighted the importance of the metabolome.” The project was called ‘The Dynamics of Plant Assimilatory Metabolism in the Post-Genome Era’ (in Japanese), and was set-up in 2000 by the Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Agency. Saito was the leader of the project before he joined PSC. In preparation for the CREST project application, Saito conducted a thorough survey of the metabolome. “For example, I even tried to use search engines on the Internet to look for information related to the metabolome or metabolomics, although only a few hits were found. This was the reality in those days. Thus, we are one of the world’s pioneers in metabolomics research.”
Why did the metabolome catch his eye ahead of other scientists? “I originally majored in pharmaceutical sciences, conducting research into sulfur-containing metabolites in plants and medicinal metabolites, such as flavonoids and alkaloids,” says Saito. “These projects were, however, considered to be individual subjects. Thus, I asked myself the question whether I could pull these subjects together to give a better understanding of plants.”
In the meantime, the whole genome sequence of Arabidopsis thaliana, a model experimental plant, was determined at the end of 2000. This also affected him greatly. “Nothing had been known about the total number of genes or metabolites before the genome sequence was clarified,” he explains. However, thanks to the genomic analysis, we understood that the number of genes is finite. Thus, I thought that the total number in the metabolome must also be finite, and that the whole world of the metabolome could be clarified.”
The prospect of vegetables containing cancer-preventive agents
What does metabolomics research help us to understand, or enable us to do? The following presents some of the recent findings by the Metabolomics Research Group.
The first finding was presented in a press release in April 2007, “Discovery of a new gene that causes the Brassica family of vegetables to produce cancer preventive agents (in Japanese).” The Brassica family of vegetables, which include calabrese, Japanese radish, Japanese horseradish, and Chinese mustard, have a piquant flavor when they are grated. The pungent components are modified metabolites called glucosinolates. Sulforaphane glucosinolate, a modified metabolite, is known to enhance neutralization of the effects of carcinogens. The Metabolomics Research Group used Arabidopsis thaliana, a member of the Brassica family of vegetables, to demonstrate that the gene PMG1 controls the production of glucosinolates.
This finding was established by using a technique called coexpression analysis combining transcriptome and metabolome data. The relationship between the expression of all Arabidopsis thaliana genes and the product yields of all metabolites was analyzed in detail using this technology, which showed a pattern in the amount of any glucosinolates throughout the Brassica family of vegetables. Thus, among the genes that follow the pattern of known glucosinolate biosynthetic genes in the Brassica family, the PMG1gene was eventually discovered (Fig. 1). This technique was established by Saito and the members of the Metabolomics Research Group. In 2004, they used the technique to discover the gene related to the metabolism and responses of sulfur in Arabidopsis thaliana. The findings were published in the Proceedings of the National Academy of Sciences of the USA (PNAS), and drew attention as the first study that successfully clarified the functions of an unknown gene by combining transcriptome and metabolome data. The impact was so great that the paper was listed in the British science magazine, Nature Biotechnology, as the most-cited paper in biotechnology in 2005.
Figure 1: An example of coexpression analysis.
Arabidopsis thaliana is estimated to have about 27,000 genes, but only one tenth of its genes are known in terms of functions. The coexpression analysis technique based on the combination of transcriptome and metabolome data is expected to clarify the functions of unknown genes. Once the functions of genes are understood, applications will come into view. “If the expression level of PMG1 is increased, and if glucosinolate, a source of sulforaphane, is produced in large quantities, we have the possibility of creating crops that have cancer-preventive effects,” says Saito. “Our collaborating company has already started research to see if the technique can be applied to vegetables.”
The second finding was presented in a press release in February 2007, “Discovery of an enzyme that determines the structure of flavonoids in plants (in Japanese).” Flavonoids are metabolites that are currently drawing attention as substances that have anticancer effects, reduce blood pressure, reduce cholesterol levels, and have antiallergic and antibacterial actions. Flavonoids need to have sugar molecules bound to their frameworks so that they can remain in existence and accumulate in a stable condition. Through coexpression analysis using Arabidopsis thaliana, the Research Group discovered a glycosyltransferase enzyme, UGT89C1, which causes sugar molecules to bind to position 7 in the framework. Without the enzyme, sugar molecules would not bind to the framework, producing different flavonoids with different structures. Different structures result in different functions. Thus the finding was a great success, suggesting the possibility of creating highly functional flavonoids.
How can Saito continue to produce such a string of significant results? “The five years under the CREST project had a profound effect. I gained much valuable experience and knowledge during that time. The scientists who worked and studied together in the same project are now playing an active role in the Metabolomics Research Group.”
Difficulties and challenges in studying metabolomics
“Our final target at PSC is to elucidate all the various possible metabolites produced by plants, and to associate them with genes, and to understand how metabolites are produced,” says Saito. “Our goal is to establish a situation in which we can positively say that this gene operation leads to the production of the metabolite you want.”
Metabolomics research, however, lags behind other areas of ome-science: the genome, the transcriptome, and the proteome. “The metabolome is several orders of magnitude more difficult,” says Saito. DNA is first transcribed into RNA. Then the sequences of the RNA are translated into amino acids. Finally the amino acids are linked together to form a protein. In this way, there is a continuity of information among the genome, transcriptome, and proteome, which allows us to estimate the relationship between them. In contrast, the metabolome refers to all the metabolites, which are only produced by enzyme proteins. “We cannot estimate the metabolome from the proteome because of the large gap between them,” says Saito.
As analysis targets, genome and transcriptome research is only concerned with nucleotides, and proteome research is only concerned with proteins, whereas metabolome research considers various kinds of metabolites including lipids, sugars, and secondary metabolites. In comparison with animals, plants have an extremely large number of metabolites. It is estimated that the number is 200,000 for the whole plant world, and about 5,000 just for Arabidopsis thaliana. For comparison, the number of metabolites is about 2,500 for human beings.
PSC has three strategies to achieve this difficult final target of metabolomics research. The first strategy is to make full use of multiple analytical instruments for metabolome analysis. These instruments are used in combination to help comprehensively identify various metabolites. For example, a mass spectrometer is combined with a gas chromatography system when the test sample can be vaporized, with a liquid chromatography system when the sample is soluble, and with a capillary electrophoresis system when the sample can be ionized (Fig. 2). Furthermore, nuclear magnetic resonance (NMR) can be used as a means to identify metabolites. However, Saito says, “We need to make efforts to further develop the technology for metabolomics research. Our technology level hardly reaches 20% of that of transcriptomics research. Thus a technical breakthrough is needed.”
Figure 2: Various instruments for metabolome analysis.
What is more important as a first strategy is how to detect temporal changes in metabolites and the resolution, because we are merely observing instantaneous profiles of metabolism. Furthermore, we cannot observe metabolic changes occurring in a cell because the whole tissue is crushed before it can be analyzed. “Ideally, we need to develop the technology available for the analysis of dynamic changes occurring in a single cell.”
The second strategy is to utilize bioinformatics. In metabolomics research, a vast amount of data has to be processed, and the volume of data is too large for experimental scientists to handle. Thus the Research Group has strong ties with experts in bioinformatics.
The third strategy is to integrate metabolomics with genome science. RIKEN has the requisite research resources, such as the mutants and over-expressors that have destroyed or over-expressed specific genes of Arabidopsis thaliana, and full-length cDNAs that contain the full information to produce specific proteins. We can deploy these resources to associate metabolites with genes.
Our aim is to develop plants that benefit mankind
How does metabolomics research proceed, and what can it bring us (Fig. 3)? Metabolomics research is now at an early stage, in which scientists are trying to understand metabolism using model experimental plants, such as Arabidopsis thaliana and the rice plant. Metabolomics research will then proceed to the second stage, in which the research results will be applied to crops, medicinal plants, and other plants that produce industrial materials such as pulp. The third stage is metabolic engineering based on our understanding of the system.
Figure 3: What metabolomics research brings about.
Many genes, proteins, and metabolites are involved in cell activities. In the future, it will become increasingly important to clarify how they interact with each other as a system. “I think metabolomics research will usher in the future,” says Saito. He continues to explain how the success of modern science is based on reductionism. For example, physics is reduced to elementary particles, chemistry, to atoms and molecules, and biology, to genes and components. They have all made great developments from these bases. In the current post-genome era, however, reductionism alone cannot provide the driving force to support further progress. “We need to understand individual elements and study the systems made up of those elements, thus going beyond the limits of reductionism. I think this will lead to an understanding of life.”
Saito also expressed his dreams for the future. “I would like to develop plant science for the benefit of mankind. I hope plant science can serve not only to provide food, drugs and medicines, and food with health functions, but also to solve environmental and energy problems, thus contributing, in a broad sense, to the health of humankind.”
(Interview and article: Shino Suzuki)