incompatible with high throughput; conventional
targeted analyses ensure reproducibility, but their
low throughput does not meet the requirements of
metabolomics. However, in recent LC-based un-
targeted analyses, high repeatability has been
achieved together with high throughput, and
reproducibility could be obtained in combination
with validation of the data for the compounds
detected. Sawada et al. ( 2009 ) proposed a widely
targeted analysis aimed at further compatibility
between throughput and reproducibility in LC-
based analysis in consideration of the data vali-
dation (Sawada et al. 2009 ). In this review, we
introduce an overview of plant metabolomics and
selected case studies ofLotus japonicus, including
targeted analyses, widely targeted analysis, inte-
grated metabolomics, an MS/MS database for
phytochemicals, and future aspects ofL. japoni-
cusmetabolomics.
16.2 Targeted Analyses
for Biosynthetic Research
on Leguminous
Phytochemicals
Fabaceae (in the Angiosperm Phylogeny Group
system) is the third most diverse family in the
flowering plants and is known as a rich source of
phytochemicals. Many of the metabolites of
members of this family have bioactivity benefi-
cial for human health (Dixon and Sumner 2003 ).
Several Fabaceae genera, such as Acacia,
Astragalus, Caesalpinia, Cassia, Glycyrrhiza,
Lablab,PuerariaandSophora,contain medici-
nal plants. Leguminous plants are also charac-
terized by symbiotic nitrogen fixation, pod
formation, metabolite accumulation in seeds and
the formation of compound leaves and bilateral
flowers. Leguminous crops are valuable resour-
ces for protein and oil; soya bean is one of the
principal world crops. Metabolomics of legumi-
nous plants is thus expected to have significant
impact on broadfields.
Of particular interest for several reasons are
flavonoids (including isoflavonoids) and triterp-
enoids in leguminous plants. In legume–rhizobia
symbiotic systems, (iso)flavonoids and
chalcones, another class of phenolic compounds
closely related toflavonoids, play essential roles
as signal molecules of the host plants towards the
symbionts (Hayashi et al. 2000 ). The isoflavo-
noid pathway of leguminous plants also produces
inducible antibiotic substances, termed phytoal-
exins, in response to challenges by biotic stresses
such as infection by pathogenic microorganisms
(Aoki et al. 2000 ). Although the distribution of
isoflavonoids is not exclusive to Fabaceae, 90 %
of isoflavonoids reported to date are found in
Fabaceae. Therefore, isoflavonoids are consid-
ered as characteristic phytochemicals of this
family. Since the 1970s, the biosynthesis of
isoflavonoid phytoalexins has been studied bio-
chemically, and the identification of 2-hydrox-
yisoflavanone synthase, the key enzyme in
isoflavone formation, from aGlycyrrhizaspecies
and soya bean (Akashi et al.1999a; Steele et al.
1999 ) has provided important clues for the
molecular basis of isoflavonoid biosynthesis.
Triterpenes are a major subgroup of the terpene
superfamily, the largest group of phytochemicals.
They are derived from the cyclization of 2,3-ox-
idosqualene by oxidosqualene cyclases (OSCs),
whosefirst identification led to subsequent studies
of this pathway (Abe and Prestwich 1995 ). Tri-
terpenes that are the direct products of OSCs, such
as α-amyrin,β-amyrin and lupeol, have been
suggested to play roles in rhizobial and mycor-
rhizal symbioses, and lupeol was shown to have
negative effects on nodule formation, as revealed
by a transgenic approach usingL. japonicus(Delis
et al. 2010 and references cited therein). Triter-
pene saponins, which are triterpenes modified by
hydroxylation and glycosylation, include various
bioactive natural products of legumes (Dixon and
Sumner 2003 ).
In general, research on plant metabolism
begins with the analysis of metabolites and the
elucidation of the condition that induces the
metabolic pathway of interest, together with a
search for suitable plant materials. Suspension
cell cultures are powerful tools in many cases.
Identification of biosynthetic enzymes includes
biochemical assays with crude extracts prepared
from the plant materials and recombinant cells
heterologously expressing cDNAs that putatively16 Metabolomics 173