recognizing only (3R)-vestitone as substrate and
not (3S)-vestitone (Shao et al. 2007 ). Amino acid
residues likely involved in the stereochemical
specificity of this VR, such as His227, are also
present in the twoL. japonicusenzymes, sug-
gesting that (3R)-vestitone is indeed their sub-
strate. Conversion of DMI to medicarpin, the
main phytoalexin ofM. sativa, is catalyzed by
DMI dehydratase (DMID) which introduces an
ether ring with the loss of water. This enzyme was
partly purified fromM. sativa, but to our knowl-
edge has not been identified at the gene level
(Guo et al. 1994 ). Conversion of medicarpin to
vestitol requires the activity of additional mem-
bers of the short-chain dehydrogenase/reductase
family. FromL. japonicusEST data, Akashi et al.
( 2006 ) were able to identify two pterocarpan
reductases, PTR1 (chr3.CM0091.1150) and
PTR2 (chr3.CM0091.1170) that converted med-
icarpin to vestitol with high-specific activity and
enantiospecificity to (-)-medicarpin.
14.5 Triterpenoids and Triterpenoid
Saponins
Triterpenoids are a diverse group of isoprene-
derived specialized metabolites often functioning
in plant chemical defense. When triterpenoids are
glycosylated, they are referred to as triterpenoid
saponins, and these amphiphatic molecules are
well known as plant chemical defense com-
pounds affecting membrane integrity (Augustin
et al. 2011 ). Oxidosqualene cyclase (OSC) is the
collective name for a group of enzymes that form
the branching point between sterol and triterpene
biosynthesis, by catalyzing alternative cycliza-
tion reactions of the common precursor 2,3-ox-
idosqualene. OSCs that function as cycloartenol
synthases lead to the production of sterols, while
other OSCs catalyze cyclization reactions that
lead to the formation of different triterpenoid
skeletons. Triterpenes suggested to occur in
L. japonicus include β-amyrin and lupeol
(Fig.14.1) (Iturbe-Ormaetxe et al. 2003 ; Sawai
et al.2006a; Delis et al. 2011 ). Saponins that are
β-amyrin derived, such as glycosides of soyas-
apogenol B, have been reported for theLotus
genus and other legumes (Tava et al. 2011 ; Golea
et al. 2012 ).
Triterpenes and their biosynthesis have been
investigated in several legume species (Seki et al.
2008 ; Tava et al. 2011 ; Carelli et al. 2011 ). In
L. japonicus, present knowledge is largely
restricted to the OSCs, the characteristic class of
branch point enzymes. OSCs fromL. japonicus
were identified by cDNA cloning and isolated
from genomic libraries and functionally charac-
terized (Iturbe-Ormaetxe et al. 2003 ; Sawai et al.
2006a). Eight OSC genes were identified that
grouped in two separate contigs located on either
chromosome 2 (contig CM0373) or chromosome
3 (contig CM0292). Functional characterization
in yeast established thatOSC1(chr3.CM0292.
500; also namedLjAMY1) encodes aβ-amyrin
synthase,OSC3(chr2.CM0373.870) encodes a
lupeol synthase, andOSC5(chr2.CM0373.850)
encodes a cycloartenol synthase (Iturbe-Orma-
etxe et al. 2003 ; Sawai et al.2006a).OSC3is
exclusively expressed in roots and nodules, and
its expression was highly induced in plants
infected with Mesorhizobium loti(Delis et al.
2011 ). OSC6 (chr2.CM0373.810) and OSC7
(chr2.CM0373.800) are closely related but dif-
ferentially expressed, either in roots and nodules
(OSC6), or in stem and leaves (OSC7). As it was
originally thought that sterols in plants are bio-
synthesized from cycloartenol and those in fungi
and animals from lanosterol, it was of particular
interest thatOSC7was able to complement a
lanosterol synthase-deficient yeast mutant (Sawai
et al.2006b). Labeling studies in Arabidopsis
have supported the existence of a plant biosyn-
thetic pathway for phytosterols via lanosterol, but
its contribution to membrane sterols was minor
in comparison with the route via cycloartenol
(Ohyama et al. 2009 ). It was suggested that the
lanosterol pathway could contribute to the pro-
duction of steroids in relation to plant defense.
LjAMY2/OSC8 (chr3.CM0292.40) is closely
related to the β-amyrin synthase OSC1, but
encodes a mixed function OSC, able to synthe-
size bothβ-amyrin and lupeol (Iturbe-Ormaetxe
et al. 2003 ). A cDNA fragment designatedOSC4
was 99 % identical withOSC8but a full-length
cDNA sequence could not be obtained, while the14 Plant-Specialized Metabolism and Its Genomic Organization... 155