The monomericflavan-3-ol units are synthe-
sized on the cytoplasmic side of the endoplasmic
reticulum and are believed to be subsequently
condensed into polymers in the vacuole (Zhao
et al. 2010 ). Thefirst committed enzyme tofla-
vonoid biosynthesis is a polyketide synthase
named chalcone synthase (CHS), which cata-
lyzes the condensation of three malonyl-CoA
molecules with one 4-coumaroyl-CoA molecule
to from naringenin chalcone. At least thirteen
CHSgenes have been identified in theL. japo-
nicusgenome, of which eight genes (CHS4- 11 )
cluster on chromosome 2 (contig CM0018, Shi-
mada et al. 2007 ). Phylogenetic analysis suggests
that this diversity ofCHSenzymes was generated
after the divergence of the legume clade and that
CHS1 (chr1.CM0104.1140) represents a non-
leguminous type. Chalcone isomerase (CHI)
catalyzes the stereo-specific cyclization of the
chalcones to form the genericflavonoid C6-C3-
C6 aromatic ring structure.L. japonicuscontains
fourCHIgenes in a 15-kb region of chromosome
5 (contig CM0180, Shimada et al. 2003 , 2007 ). It
is of interest to note that while CHI1, CHI3, and
CHI4 are close homologs that are 90 % identical
at the amino acid sequence level, CHI2 only
showed 50 % amino acid identity with the other
three lotus enzymes. CHI2 is similar to non-
leguminous CHIs (referred to as type I) and
catalyzes the isomerization of 6′hydroxychalcone
to the 5′-hydroxyflavone naringenin, which is a
precursor of allflavonoids including theflavan-
3-ol units of PAs. CHI1 and CHI3 are more
similar to legume-specific CHIs (referred to as
type II), which in addition to the type I reaction
also convert 6′-deoxychalcone to 5′-deoxyflav-
one, the precursor for isoflavonoid biosynthesis.
The next step is the hydroxylation of the C-ring
by the non-heme iron enzyme flavanone 3-
hydroxylase (F3H) to give the dihydrokaempfer-
ol. The cytochrome P450 enzymeflavonoid 3′-
hydroxylase (F3′H) catalyzes the hydroxylation at
the 3 position of the B-ring to form dihydroqu-
ercetin. This is a precursor for the procyanidin
flavan-3-ol units that will be incorporated into
PAs. A second cytochrome P450,flavonoid 3′-5′-
hydroxylase (F3′ 5 ′H), catalyzes the hydroxylation
of dihydroquercetin at the 5 position of the B-ring
to form dihydromyricetin. Conversely, this is the
precursor for the prodelphinidinflavan-3-ol units
of PAs. Genes encoding F3H, F3′H, and F3′ 5 ′H
enzymes have not been investigated in any detail
inL. japonicus, but candidate genes can be readily
identified in its genome sequence. The subsequent
reduction of the various B-ring hydroxylated
intermediates to leucoanthocyanidins is catalyzed
by a reductase–epimerase–dehydrogenase (RED)
super family member, the enzyme dihydroflavo-
nol reductase (DFR). This is thefirst committed
enzyme of the anthocyanin and PA branch of the
flavonoid pathway. Five DFR genes that form a
cluster in a 38-kb region on chromosome 5 (on
contig CM0077) were previously characterized
(Shimada et al. 2005 ). The encoded DFRs have
alternative substrate preferences for the various B-
ring hydroxylated dihyroflavonols, and variation
in the expression of these genes inLotusspecies
could explain the differences in hydroxylation
patterns of PA units, but additional regulatory
mechanisms are also thought to exist (Shimada
et al. 2005 ). It was more recently shown that only
theDFR2promoter is activated by the transcrip-
tion factor LjTT2, a known MYB regulator of PA
biosynthesis genes (Yoshida et al. 2010 ).
The leucoanthocyanidins are an intermediate
in anthocyanin biosynthesis and also a substrate
for thefirst specific step for PA biosynthesis. It is
speculated that they may also be used as exten-
sion units of PA polymers (Pang et al. 2007 ).
Their reduction to 2,3-trans-flavan-3-ols such as
catechin, the typicalL. japonicusPA terminal
unit, is catalyzed by leucoanthocyanidin reduc-
tase (LAR). Like DFR, LAR is a member of the
RED super family but is more closely related to
isoflavone reductase-like proteins than DFR. Two
cDNAs encoding putative LARs have been
cloned fromL. corniculatus, both of which are
expressed in leaf tissue (Paolocci et al. 2007 ).
When both types of LcLARs were expressed in
E. coli,they were able to catalyze the formation of
catechin. InL. japonicus,the gene corresponding
toLcLAR1is probably Chr2.CM0124.20 and a
DNA fragment encoding a similar sequence to
LcLAR2is also present (LjSGA_076819.1).14 Plant-Specialized Metabolism and Its Genomic Organization... 151