Omics analysis nurtures the creation of functional plants with clinical applications.

Plants produce a wide variety of metabolites from inorganic compounds,
some with useful functions including health-promoting effects. The ability
to harness these metabolites by creating ‘functional plants’ that produce
these compounds in large quantities could therefore be of considerable benefit
to society. It is also an intriguing research topic for plant scientists. Progress
in this field relies on our understanding of how the hundreds of thousands
of unique metabolite compounds are produced in plants. Masami Yokota
Hirai at the RIKEN Plant Science Center (PSC) is investigating the mechanism
of metabolism in plants by linking metabolites to genes through omics
analysis—a combination of metabolome and transcriptome analyses. The
knowledge obtained from this approach is leading steadily to the successful
development of functional plants.
Compound synthesis by metabolism in plants
The word ‘metabolism’ may remind many people of a reaction in which the proteins or lipids
we consume as food are degraded and converted into energy. Metabolism in plants,
however, is different from this reaction,” says Masami Yokota Hirai, team leader of the
Metabolic Systems Research Team at the RIKEN Plant Science Center (PSC). “Metabolism
in plants is a reaction in which inorganic compounds such as nitrogen, phosphorus, and
sulfur are absorbed through the roots and light energy is used to produce various organic
compounds including amino acids and sugars such as starch. These organic compounds
are called metabolites.”


The production of compounds such as amino acids, sugars and vitamins, which make up the body
of a plant, from inorganic compounds is called ‘primary metabolism’, whereas the production of
more complex compounds from primary metabolites is called ‘secondary metabolism’. “A plant is
rooted in place and cannot move. To cope with environmental hazards, such as insects, dry
weather or salt damage, plants produce secondary metabolites as they are exposed to these
stresses. A plant maintains a constant amount of primary metabolites, but produces secondary
metabolites on an as-needed basis.”
Hirai’s focus on metabolism in plants is motivated by the potential uses of the vast array of metabolites
that plants produce. “Plants produce metabolites that specific to the plant species. There are more
than 200,000 known unique metabolites, some of which contain nutrient, health-promoting and
medical ingredients.” Examples of secondary metabolites include isoflavone, anthocyanin, menthol,
catechin and capsaicin (Fig. 1). These compounds are drawing attention for their health-promoting
functions. “Efficient production of useful secondary metabolites will greatly help improve our health.
For this reason, we are working hard to elucidate the metabolic mechanism in plants.”
Metabolome studies
“A metabolic map illustrates how metabolites are produced from inorganic substances through
specific reactions, and can be as complicated as a subway route map. A partial metabolic map
does not provide enough information to understand the whole picture of metabolism. We need
to understand the metabolic system as a whole. To help with this, we have started to take advantage
of omics analysis.”
Omics is a science that comprehensively embraces the four disciplines of genomics, transcriptomics,
proteomics and metabolomics, and has developed rapidly in the past decade triggered by
improvements in genome decoding techniques and processing speed. These improvements have
led to some remarkable milestones in genomic research, including sequencing of the complete genomes
of the flowering plant Arabidopsis thaliana, rice and soy-bean.


Similarly, the DNA microarray technique used in transcriptome analysis, the analysis of RNA
transcribed from DNA, has also improved dramatically over the past decade. In this technique,
hundreds of thousands of single-stranded DNA fragments are fixed in holes or ‘spots’ on a glass
substrate, and fluorescently labeled RNA are dropped onto the substrate surface. RNA
complementary to a DNA fragment will become bound to the DNA, which causes the combined
compound to emit fluorescent light. From the fluorescence intensity of each spot, researchers
can determine which genes are being expressed and to what extent. The expression of Arabidopsis
genes has been analyzed using this method and the data has been made publicly available via
the At GenExpress database.

Progress in metabolome analysis, however, lags considerably behind that of genomic and
transcriptome analyses. “Metabolome analysis is the least developed part of omics,” says Hirai.
“In metabolome analysis, a mass spectrometer is used to determine the mass of molecules
and electrical charges contained in a specimen. From this data we can determine the types
and quantities of metabolites in a specimen. The work, however, is extremely difficult.
Genomics focuses only on DNA and transcriptomics on RNA, and the same method can
be used for all types of organism. In metabolomics, on the other hand, we deal with
metabolites with a wide range of characteristics, such as volatility and water solubility,
which makes it impossible to conduct investigations under fixed conditions. The
metabolites are also produced in highly variable amounts, tiny to large quantities,
and with a wide range of concentrations. So we need to share a single specimen
among a number of measuring instruments so that enough data can be collected.”
The DNA microarray technique is almost entirely automated, which allows almost
anybody to use it, whereas mass spectroscopy requires a highly skilled operator.
This has obstructed rapid developments in metabolome analysis. Another factor
lies with the collected data itself. “The data obtained by mass spectrometry are
plotted on a graph with mass along the horizontal axis and intensity along the
vertical axis. A peak in the graph corresponds to a single metabolite. Of the
thousand or so peaks that are produced, only about 10% have been assigned
to specific metabolites. Most of the metabolites remain unknown. We are at a
loss regarding where to begin,” says Hirai.
Research into metabolomics started in 2000. In Japan, Kazuki Saito, group
director of the Metabolomic Function Research Group at the PSC, took the
initiative in research on metabolomics. In those days, Saito was a professor
at the Graduate School of Pharmaceutical Sciences at Chiba University, and
Hirai attended his laboratory. “I was confident that the research was not only
interesting but also very important. However, I had a hard time for about three
years because I could not find a way to understand the data itself,” says Hirai,

The world’s first omics analysis
In 2004, Hirai published a paper on metabolome analysis in the Proceedings
of the National Academy of Sciences, USA. The article would become the
most-cited paper in the field of plant biotechnology in 2005. “This paper is a
collection of results obtained from an integrated analysis of the transcriptome
and metabolome of A. thaliana. The paper does not provide new information
on gene functions or metabolite synthesis, so I am not completely satisfied with
the paper because it is a simple description of my research results. But in those
days, few papers could be found on metabolome analysis. I think that the paper
was highly evaluated under such circumstances because I tried to derive
something new by combining transcriptome analysis with metabolome analysis
for Arabidopsis. It was a pioneering attempt at omics analysis.”
Investigation of all 27,000 Arabidopsis genes shows that there are multiple genes
that express with the same timing. These genes are likely to be involved in the same
function. If the population of a metabolite increases while a certain gene cluster
is expressing and decreases when the gene cluster is not expressing, the metabolite
could be associated with the gene cluster. In this way, omics analysis allows
genes to be linked with metabolites, making it possible to understand metabolite
One successful application of omics analysis is the 2007 discovery of a new gene
that makes cruciferous vegetables produce cancer-preventing components.
Cruciferous vegetables such as broccoli, radish, horse radish and mustard have
a ‘spicy’ flavor that has been attributed to pungent components that originate as
metabolites called glucosinolates, among which sulforaphane is known to enhance
the functions of enzymes that detoxify carcinogens. Using Arabidopsis, a member
of the cruciferous family, Hirai successfully showed that the gene PMG1
controls the synthesis of glucosinolates.
“Through an integrated analysis of the transcriptome and metabolome of Arabidopsis,
we found a gene cluster that changed with the same pattern. The gene cluster was
found to contain the genes that are known to be involved in the synthesis of glucosinolates.
In this way, we looked into the genes, and finally reached PMG1.”
It was also confirmed that the functional enhancement of PMG1 in Arabidopsis promotes
glucosinolate synthesis. “As the amount of glucosinolate increases, the amount of
sulforaphane increases. This could allow us to grow vegetables with enhanced
cancer-preventing effects.”
A study focusing on gene clusters associated with the synthesis of glucosinolates
is now under way. Hirai is interested in the gene BASS5. The base sequence of
BASS5 is similar to that of the genes for bile acid transporter, an animal protein. Bile
acid transporter is present in the cell membrane and is responsible for the intercellular
movement of bile acids. When the base sequences are similar, their functions are
also often similar. But since there are no bile acids in plants, the role of the proteins
created by BASS5 is particularly interesting. “The proteins were thought at first to be
present in the cell membrane where they mediate the intercellular movement of
glucosinolates. However, we have come to understand that the proteins are likely
to be present not in the cell membrane but on the surface of the cell’s chloroplast.
This demonstrates that secondary metabolites are synthesized not only in the cellular
cytoplasm, but also in the chloroplasts. We think BASS5 might be associated with
glucosinolate intermediates moving in and out of the chloroplasts.” By omics analysis
it was confirmed that glucosinolates are not created when BASS5 function is
inhibited. Omics analysis has been demonstrated in this way time and time again
to be a very effective tool for elucidating metabolic pathways.
Hirai is also researching the synthesis of amino acids, particularly methionine, the
primary metabolite from which glucosinolates are produced. “An increase in a metabolite
requires an increase in the supply of its source material, namely amino acids. We
should know how to synthesize amino acids if we are to attempt to create plants
that produce large amounts of useful secondary metabolites.”
Metabolome analysis in full swing
“Metabolome analysis will proceed rapidly in the years to come,” says Hirai with
confidence. This is thanks to an analytical technique called widely targeted metabolome
analysis developed by Yuji Sawada, a special research scientist in the Metabolic
Systems Research Team. Associating the thousands of peaks produced by mass
spectrometry with metabolites has been one of the obstacles to metabolome research.
“In conventional targeted metabolomics, observations are made with the aim of
identifying a single known kind of metabolite,” says Hirai. “Widely targeted metabolomics
is based on the idea that if the number of target metabolites can be increased,
eventually all metabolites could be targeted, which could lead to full metabolome
analysis. In widely targeted metabolomics, all of the peaks in the data correspond
to known metabolites. This allows us to proceed to the next stage of the research
program immediately. Usually, analytical methods are developed by analytical
chemists and information scientists, but Dr Sawada and myself are biologists.
Widely targeted metabolomics is a very convenient analytical method for biologists.
” Widely targeted metabolomics currently handles about 700 target metabolites, a
number that will be increased in the near future.
Hirai also hopes to develop a new analytical method for omics analysis. “It is
essential to develop additional tools if we are to remain pioneers in this field
because integrated analysis of transcriptome and metabolome data is now available
to everyone. We are now working on developing, by trial and error, a new analytical
method that can suggest unforeseen results.”