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718 Plant Mol Biol (2(106) 62:715-733
(3), which undergoes dehydration to yield the isoflav-
one formononetin (4) (Fig. 1A). Formononetin accu-
mulates in several legumes and is also a precursor in
the biosynthesis of medicarpin (5) and related ptero-
carpanoid phytoalexins. HI4'OMT is closely related to
PsHMM at the amino acid sequence level (Akashi
et al. 2003), and has been shown to possess 6a-hy-
droxymaackiain 3-OMT activity (Liu et al. 2005).
On the basis of transgenic studies implicating
MsI7OMT in the formation of 4'-O-methylated isofi-
avonoids in alfalfa (He and Dixon 2000), structural
studies suggesting that 2-hydroxyisoflavanone can be
accommodated in the active site of MsI7OMT in an
orientation favorable for 4'-O-methylation (Zubieta
et al. 2001), and cellular imaging studies indicating
potential association of the operationally soluble
MsI7OMT with IFS on the outer surface of the endo-
plasmic reticulum (Liu and Dixon 2001), we proposed
that the substrate specificity of MsI7OMT may differ in
vivo (B- ring 4'-position) from its in vitro specificity
(A-ring 7-position). The discovery of a 2-hydroxyisof-
lavanone-specific 4'-OMT (Akashi et al. 2003) calls
into question this earlier interpretation of MsI7OMT's
role in 4'-O-methylation.
Expressed sequence tag (EST) libraries obtained
from the model legume Medicago truncatula contain
several related OMT sequences with homology to
I7OMT or H14'-OMT, and therefore annotated, on the
basis of sequence identity, as encoding isoflavone 7-
OMTs, 2-hydroxyisoflavanone 4'-OMTs, or 6a-hy-
droxymaackiain OMTs. To better understand the nat-
ure of the OMTs catalyzing the 4' and 7-O-methylation
reactions during isoflavonoid biosynthesis in Medicago,
and to provide experimental evidence for the tentative
annotations based on sequence similarity, we cloned,
heterologously expressed and determined relative
specificities for (iso)flavonoid substrates of seven
putative M. truncatula IOMTs (MtIOMTs).
Materials and methods
Cloning, expression, and purification of
M. truncatula IOMTs
Candidate M. truncatula IOMTs were identified by
searching MtGI Release 7.0 (May 1, 2003) (http://
www.tigr.org/tigr-scripts/tgi/T_index.cgi?species = me-
dicago) for ESTs with similarity to M. sativa I7OMT
(GB accession number U97125) using BLAST sear-
ches. Full-length ESTs for MtIOMTI (NF070-
A11EC1F1082) were obtained from an elicited
M. truncatula cell culture library; full length MtIOMT3
ESTs (NF027F03PLlF1029) were obtained from a
phosphate-starved leaf library; full length MtIOMT4
ESTs (N201058e) were obtained from a phosphate-
starved root library; full length MtIOMT6 (NF031-
D06RTIF1046), MtIOMT7 (NFOO5H09RTlF1079),
and MtIOMT8 ESTs (NF031H02RTlF1016) were ob-
tained from a M. truncatula developing root library.
Cloning of MtIOMT5 (corresponding to TC 100926,
TIGR MtGI v.8) was as described previously (Liu
et al. 2005). To obtain a full-length cDNA for MtI-
OMT2, specific primers designed based upon the
M. truncatula genomic sequence were used to amplify
the MtIOMT2 coding sequence from M. truncaula
root cDNA. The amplified product was cloned into the
pGEM-T easy vector (Promega, Madison WI) and
sequenced to confirm its identity. MtIOMTs were
cloned into the pET28a vector (EMD Biosciences, Inc.,
San Diego, CA) using PCR-based amplification with
primers designed to introduce EcoRI and XhoI
restriction sites; BamHI and Xhol sites were used for
cloning MtIOMT6 due to the presence of an internal
EcoRI site in this particular cDNA. Because the EST
for MtIOMT8 was missing a start codon, the primer
designed to clone MtIOMT8 into pET28a contained an
additional 2-nucleotide insertion to generate an ATG
start site. All MtIOMTs were expressed as transla-
tional fusions to an N-terminal His-tag. Expression of
MtIOMTs in E. coli BL21(DE3) and purification by
Nit+-NTA chromatography were as described (Zubieta
et al. 2001). Proteins were dialyzed against 20 mM Tris
pH 8.0, 100 mM NaCl, 10% (v/v) glycerol, and 14 mM
#-mercaptoethanol. Protein concentration was deter-
mined by Bradford assay using BSA as a standard
(Bradford 1976).
OMT assays
Reactions (200 pl) were performed in 0.1 M potassium
phosphate, pH 7.4, 10% (w/v) sucrose, 14 mM fl-mer-
captoethanol with 10 pM protein, 0.5 mM S-adenosyl-
L-methionine, and 80 pM phenolic substrates. All
substrates were purchased from Indofine (Hillsbor-
ough, NJ) except 6,7-dihydroxy-4'-methoxyisoflavone.
dihydrodaidzein, and vestitol, which were purchased
from Apin (Oxfordshire, UK). 2,7,4'-Trihydroxyisof-
lavanone was purified from IFS reactions carried out
with liquiritigenin as described (Liu et al. 2005). OMT
assays were incubated at 30C for 2 h and extracted
with ethyl acetate. Ethyl acetate extracts were dried
under N2 and the resultant material was resuspended
in 50-100 l of methanol. Samples were analyzed on
an Agilent 1100 HPLC, equipped with a quaternary
pump (model # G1311A), a degasser (model #
' Springer
718
Plant Mol Biel (2006) 62:715--733
