lotaustralin and linamarin (Takos et al. 2011 ).
The biosynthetic pathway was further supported
by genetic evidence. The absence of all
hydroxynitrile glucosides in thecyd1mutant in
CYP79D3demonstrated its inability to produce
the shared oxime intermediates. A strong reduc-
tion in the cyanogenic glucosides lotaustralin and
linamarin was observed in thecyd4mutant in
CYP736A2. Due to the specificity of CYP736A2,
no rhodiocyanosides were produced in the
above-mentioned transient expression in tobacco,
and rhodiocyanoside A and D levels were not
reduced in the cyd4mutant. A genetic locus
namedRhois responsible for the production of
rhodiocyanosides but also contributes to cyano-
genic glucoside production.Rhois closely linked
to the gene cluster but falls outside the presently
available sequence of the CM0241 contig (Takos
et al. 2011 ). Biochemical evidence suggested the
involvement of a cytochrome P450 enzyme to
form 2-methyl-2-butenenitrile as an intermediate
in the biosynthesis of rhodiocyanoside A,
requiring a subsequent hydroxylation step to
form the rhodiocyanoside A aglycone (Saito
et al. 2012 ). The biosynthetic genes responsible
for these steps are being identified.
14.7 Biosynthetic Gene Clusters
inL. Japonicus
It has become apparent that the pathways for
several classes of chemical defense compounds
are clustering in plant genomes (Chu et al. 2011 ;
Takos and Rook 2012 ). It is important to stress
that these gene clusters are not just repeats of
homologous genes, which are more common as
illustrated by many of the gene families in phe-
nylpropanoid metabolism described earlier, but
consist of non-homologous genes encoding dif-
ferent types of enzymes of the same biosynthetic
pathway. Such biosynthetic gene clusters have
been described for other plant chemical defense
compounds such as for DIBOA (2,4-dihydroxy-
1,4-benzoxazin-3-one) in maize (Frey et al.
1997 ), the triterpenoids avenacin in oat, and
thalianol and marneral in Arabidopsis (Qi et al.
2004 ; Field et al. 2008; Field et al. 2011 ),
diterpenoid phytoalexins such as momilactones
and phytocassanes in rice (Wilderman et al.
2004 ; Shimura et al. 2007 ; Swaminathan et al.
2009 ), the alkaloid noscapine in opium poppy
(Winzer et al. 2012 ), and recently for terpene
biosynthesis and steroidal glycoalkaloids in
solanaceous species such as tomato (Matsuba
et al. 2013 ; Itkin et al. 2013 ). ForL. japonicus,
we previously reported a gene cluster for the
biosynthesis of cyanogenic glucosides (Fig.14.3)
consisting of the genesCYP79D3, CYP79D4,
CYP736A2,andUGT85K3, also demonstrating
that the analysis of a gene cluster can aid gene
discovery (Takos et al. 2011 ).
Several gene clusters have so far been reported
for triterpenoid biosynthetic pathways. Their
positive identification benefits from the charac-
teristic presence of oxidosqualene cyclase genes.
OSCs catalyze the cyclization of the common
precursor 2,3-oxidosqualene, representing the
first committed step of the pathway (Augustin
et al. 2011 ). Various modifications to the back-
bone structure are catalyzed by enzymes such as
cytochrome P450 s which have a broad functional
diversity. Complex glycosylation patterns of tri-
terpenoid saponins are made by the activity of
UDP-glycosyltransferases. Both these enzyme
classes are encoded by large gene families, and
assigning specific metabolic functions to indi-
vidual genes requires biochemical and genetic
evidence. The existence of a putative gene cluster
would immediately suggest candidate genes for
further analysis. The genomic region containing
OSC1andOSC8on contig CM0292 seems to
contain a triterpenoid gene cluster, and a func-
tional characterization of one cytochrome P450
gene in this region was recently reported
(Fig.14.3; Krokida et al. 2013 ). TheCYP71D353
gene (chr3.CM0292.110) encodes a cytochrome
P450 enzyme proposed to catalyze the oxidation
of 20-hydroxylupeol to 20-hydroxybetulinic acid.
The adjacent chr3.CM0292.120 encodes
CYP88D5, and chr3.CM0292.180 is identical to
CYP88D4. Members of the CYP88D subfamily
were previously reported to be involved in tri-
terpene biosynthesis, for example, CYP88D6
functions as aβ-amyrin C11-oxidase inGlycyr-
rhiza uralensis(Seki et al. 2008 ). A complete and14 Plant-Specialized Metabolism and Its Genomic Organization... 157