sequence of a genomic clone containing it was
unclear (Sawai et al. 2006a). In the present
assembly build 2.5 of theL. japonicusgenome,
OSC4 likely corresponds to the partial gene
sequence chr3.CM0292.280, while OSC2is a
pseudogene located between OSC4and OSC1
(Sawai et al.2006a). Identification of further
triterpenoid biosynthetic genes inL. japonicusis
proposed to benefit from the existence of a bio-
synthetic gene cluster in the region containing
OSC1andOSC8, as will be discussed below.
14.6 Hydroxynitrile Glucosides
and Cyanogenesis
inL. japonicus
A prominent class of chemical defense com-
pounds in legumes is that of the hydroxynitrile
glucosides, of which the α-hydroxynitrile glu-
cosides are commonly referred to as cyanogenic
glycosides. Cyanogenic glucosides are part of a
constitutive two-component plant chemical
defense system. Upon tissue damage by herbi-
vore feeding, cyanogenic glucosides are hydro-
lyzed by specific β-glucosidase enzymes,
releasing the unstableα-hydroxynitrile aglycone
which dissociates into toxic hydrogen cyanide
(HCN) and a ketone or aldehyde. The experi-
mental resources available inL. japonicushave
promoted this species as the genetic model to
study hydroxynitrile glucoside metabolism and
its evolution, and a large number ofcyanogenesis
deficient mutants (cyd) were identified in a
genetic screen (Takos et al. 2010 , 2011 ).
L. japonicuscontains the cyanogenic gluco-
sides lotaustralin and linamarin (Forslund et al.
2004 ). In addition, it contains the non-cyanogenic
γ-hydroxynitrile glucoside rhodiocyanoside A and
theβ-hydroxynitrile glucoside rhodiocyanoside D
(Fig.14.1). Lotaustralin and rhodiocyanoside A
are the two major hydroxynitrile glucosides in the
L. japonicus accessions MG20 and Gifu, but
intraspecific variation in hydroxynitrile glucoside
composition has been documented, for instance,
the absence of rhodiocyanosides in the MG74
accession ofL. japonicus(Bjarnholt et al. 2008 ).
A role for rhodiocyanoside A in chemical defense
has been more difficult to establish, but a toxic
furanone product is formed following its hydro-
lysis (Bjarnholt and Møller 2008 ; Saito et al.
2012 ).
The biosynthesis of hydroxynitrile glucosides
starts from amino acids with linamarin derived
from valine and lotaustralin and the rhodiocy-
anosides derived from isoleucine. The biosyn-
thesis of linamarin and lotaustralin involves three
enzymatic steps, catalyzed by two cytochrome
P450 enzymes and a glucosyltransferase acting
sequentially. The close paralogsCYP79D3(chr3.
CM0241.700) and CYP79D4 (chr3.CM0241.
310) encode the enzymes for thefirst biosyn-
thetic step of the pathway, converting isoleucine
or valine into their corresponding oximes. The
genes differ in their expression pattern, with
CYP79D3highly expressed in newly developing
leaves and involved in leaf cyanogenesis, and
CYP79D4 expressed in roots (Forslund et al.
2004 ). The oxime produced from isoleucine is
not only thefirst intermediate in the biosynthesis
of lotaustralin but also in the synthesis of rhod-
iocyanoside A and D. The biosynthetic pathway
for lotaustralin and rhodiocyanosides diverges at
the second enzymatic step, the formation of
the hydroxynitrile aglycones. Identification of the
enzyme for this second step inL. japonicuswas
made possible by the observation that all bio-
synthetic genes for the synthesis of cyanogenic
glucosides co-localized in the genome (Takos
et al. 2011 ). We also observed such gene clusters
for cyanogenic glucoside biosynthesis in the
genomes of cassava (Manihot esculenta) and
sorghum (Sorghum bicolor). Unlike in these last
two cyanogenic species, the second step in
L. japonicusdid not involve a member of the
CYP71 family of cytochrome P450 enzymes, but
was catalyzed by the product of theCYP736A2
gene (chr3.CM0241.850) present in the gene
cluster. The gene encoding the final enzyme
responsible for glucosylation and stabilization of
the hydroxynitrile aglycone, the UDP-glucosyl-
transferase geneUGT85K3(chr3.CM0241.610),
is also part of the gene cluster. Transient co-
expressing of CYP79D3, CYP736A2, and
UGT85K3inNicotiana benthamianaresulted in
the production of the cyanogenic glucosides156 A.M. Takos and F. Rook