Genomic and Coexpression Analyses Predict Multiple Genes
Transcript of Genomic and Coexpression Analyses Predict Multiple Genes
Genomic and Coexpression Analyses Predict MultipleGenes Involved in Triterpene Saponin Biosynthesis inMedicago truncatula C W
Marina A. Naoumkina, Luzia V. Modolo,1 David V. Huhman, Ewa Urbanczyk-Wochniak, Yuhong Tang,
Lloyd W. Sumner, and Richard A. Dixon2
Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401
Saponins, an important group of bioactive plant natural products, are glycosides of triterpenoid or steroidal aglycones
(sapogenins). Saponins possess many biological activities, including conferring potential health benefits for humans.
However, most of the steps specific for the biosynthesis of triterpene saponins remain uncharacterized at the molecular
level. Here, we use comprehensive gene expression clustering analysis to identify candidate genes involved in the
elaboration, hydroxylation, and glycosylation of the triterpene skeleton in the model legume Medicago truncatula. Four
candidate uridine diphosphate glycosyltransferases were expressed in Escherichia coli, one of which (UGT73F3) showed
specificity for multiple sapogenins and was confirmed to glucosylate hederagenin at the C28 position. Genetic loss-of-
function studies in M. truncatula confirmed the in vivo function of UGT73F3 in saponin biosynthesis. This report provides a
basis for future studies to define genetically the roles of multiple cytochromes P450 and glycosyltransferases in triterpene
saponin biosynthesis in Medicago.
INTRODUCTION
Interest in plant natural products has recently increased due to
the realization of their importance for animal and human health.
Saponins, a group of natural products widespread throughout
the plant kingdom, are glycosides of triterpenoid or steroidal
aglycones (Abe et al., 1993; Osbourn, 2003; Vincken et al., 2007),
and the corresponding aglycones are termed sapogenins. The
name saponin is derived from the Latin word “sapo,” indicating
that the plant contains a frothing agent when its extract is mixed
with water. The foaming ability of saponins is caused by the
combination of the hydrophobic sapogenin with the hydrophilic
sugar substituent(s).
Triterpenoid saponins are widely found in the Leguminosae
(Huhman and Sumner, 2002; Dixon and Sumner, 2003; Suzuki
et al., 2005). Their biological activities can positively or negatively
impact plant traits (Dixon and Sumner, 2003). For example,
saponins confer protective functions to the plant due to their
antimicrobial, antifungal, anti-insect, and antipalatability activi-
ties (Kendall and Leath, 1976; Tava andOdoardi, 1996; Osbourn,
2003) but can be toxic tomonogastric animals and reduce forage
digestibility in ruminants (Oleszek, 1996; Small, 1996; Oleszek
et al., 1999). They also have potential health benefits for humans,
and plants containing saponins have long been used in tradi-
tional medicine. Recent studies have illustrated useful pharma-
cological properties of saponins, including anticholesterolemic
and anticancer activities (Waller and Yamasaki, 1996; Behboudi
et al., 1999; Haridas et al., 2001; Chen et al., 2005). Saponin-
based adjuvants have the unique ability to enhance immunity
(Rajput et al., 2007). In addition to cosmetic and pharmaceutical
products, saponins have also found wide applications in bever-
ages and confectionery (Price et al., 1987; Petit et al., 1995;
Uematsu et al., 2000; Sparg et al., 2004).
Most of the steps in the biosynthesis of triterpene saponins
remain uncharacterized at the molecular level (Haralampidis
et al., 2002; Dixon and Sumner, 2003). Triterpenoid saponins
are synthesized via the isoprenoid pathway by cyclization of
2,3-oxidosqualene to yield the triterpenoid skeleton of b-amyrin
(Haralampidis et al., 2002). This step is catalyzed by a specific
cyclase, b-amyrin synthase (b-AS), which has been functionally
characterized from several plants, including Arabidopsis thaliana
(Shibuya et al., 2008), licorice (Glycyrrhiza echinata; Hayashi
et al., 2001), oat (Avena sativa; Qi et al., 2004),Saponaria vaccaria
(Caryophyllaceae; Meesapyodsuk et al., 2007), garden pea
(Pisum sativum; Morita et al., 2000), and the model legume barrel
medic (Medicago truncatula; Suzuki et al., 2002). However, little
progress has been made in characterization of the enzymes
involved in modification of the triterpenoid backbone; these
include cytochrome P450-dependent monooxygenases, uridine
diphosphate glycosyltransferases (UGTs), and occasionally
other enzymes (Haralampidis et al., 2002).
Functional genomics approaches are powerful tools to facil-
itate the understanding of secondary metabolism in plants.
For example, amplified fragment length polymorphism-based
1Current address: Departamento de Botanica, Instituto de CienciasBiologicas, Universidade Federal de Minas Gerais, Av. Antonio Carlos6627, Pampulha, Belo Horizonte, MG 31270-901, Brazil.2 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Richard A. Dixon([email protected]).CSome figures in this article are displayed in color online but in blackand white in the print edition.WOnline version contains Web-only datawww.plantcell.org/cgi/doi/10.1105/tpc.109.073270
The Plant Cell, Vol. 22: 850–866, March 2010, www.plantcell.org ã 2010 American Society of Plant Biologists
transcript profiling in combination with targeted metabolite anal-
ysis has been applied for the discovery of genes involved in
secondary metabolism in tobacco (Nicotiana tabacum) cells
(Goossens et al., 2003), and an integrated approach coupling
transcriptome coexpression analysis with reverse genetics
has been used for functional identification of members of a
multigene family of flavonoid glycosyltransferases inArabidopsis
(Yonekura-Sakakibara et al., 2007). Genes encoding enzymes
functioning in secondary metabolism are generally more diver-
gent than those involved in primary metabolism. Although genes
for most metabolic pathways in plants are not organized in gene
clusters, a small but increasing number of operon-like gene
clusters have been identified for synthesis of plant defense
compounds (Frey et al., 1997; Qi et al., 2004; Wilderman et al.,
2004; Shimura et al., 2007; Field and Osbourn, 2008; Osbourn
and Field, 2009). Investigation of the genomic organization of
metabolic pathways may therefore be a useful additional ap-
proach to ascribing potential function to candidate pathway
genes as well as shedding further light on the evolution of
chemical diversification in plants.
Root-derived cell suspension cultures of M. truncatula accu-
mulate triterpene saponins after exposure to the wound signal
methyl jasmonate (MJ) (Suzuki et al., 2005). Here, we use
comprehensive clustering of MJ-induced transcript expression
patterns, along with chromosomal location analysis, as tools to
understand triterpene saponin biosynthesis, in particular, the
potential involvement of specific P450 and UGT genes. As proof
of concept, we expressed four candidate M. truncatula UGTs in
Escherichia coli, one of which showed specificity for multiple
sapogenins in vitro and was confirmed to be involved in saponin
biosynthesis in vivo through genetic loss-of-function analysis.
Such genes may have potential for improving the quality of
forage legumes through metabolic engineering.
RESULTS
Ontology of Differentially Regulated Genes inM. truncatula
Cell Cultures
Exposure ofM. truncatula cell suspension cultures to MJ results
in a strong increase in triterpene saponin levels (Suzuki et al.,
2005). Transcriptional changes in response to the pathogen
mimic yeast elicitor (YE) occur early (maximal at 2 h after
elicitation), whereas the majority of gene expression changes in
response to MJ occur later (maximal at 24 h after elicitation)
(Suzuki et al., 2005; Naoumkina et al., 2007). Affymetrix micro-
array analysis was therefore performed to identify transcript
changes in elicited cells at these two critical time points, with
corresponding controls.
From 50,900 M. truncatula probe sets represented on the
Affymetrix array, 7836 passed a statistical significance test
(adjusted for false discovery rate) for being differentially ex-
pressed in response to YE or MJ. We applied the M. truncatula
MedicCyc database (Urbanczyk-Wochniak and Sumner, 2007)
to place the distribution of differentially expressed probe sets
into functional categories (excluding those with unidentified
function). In this database, nonplant pathways are excluded,
and plant/legume-specific pathways such as isoflavonoid, triter-
pene saponin, and lignin are featured in detail. The frequencies of
the groups of genes that are over represented in the upregulated
and downregulated probe set categorieswere compared relative
to the frequencies at which they occur on the microarray. More
probe sets of all functional classes were upregulated at 2 h after
YE elicitation, and more probe sets were downregulated at 24 h
after MJ elicitation (see Supplemental Figure 1 online). Tran-
scripts encoding genes classified in the secondary metabolism,
signaling, miscellaneous enzyme, hormone metabolism, stress,
and lipid metabolism categories were overrepresented among
the upregulated probe sets, whereas genes classified in cell wall
metabolism, signaling, and polyamine metabolism were over-
represented among those downregulated. Notably, the relative
abundance of genes classified in secondary metabolism was
5 times higher among the upregulated probe sets than among
the downregulated probe sets.
TranscriptionalReprogrammingandGenomicOrganization
of Core Terpene Pathway Genes
Of the more than 30 different triterpene saponins detected in M.
truncatula cell suspension cultures exposed to YE or MJ, many
are strongly induced only by MJ (Suzuki et al., 2005). Such
differential accumulation of saponins was reflected by differ-
ences in gene expression levels in the YE andMJ Affymetrix data
sets (Figure 1; see Supplemental Data Set 1 online). These
changes start with the primary metabolic pathways that feed into
triterpene biosynthesis.
The first steps of the acetate-mevalonate pathway to terpenes
lead to the formation of isopentenyl diphosphate (IPP) and
dimethylallyl diphosphate (Lange andGhassemian, 2003) (Figure
1). Thiolase and HMG-CoA synthase probe sets were upregu-
lated 2 to 4 times byMJ, but not by YE. Of the two thiolase genes
in the M. truncatula genome, Medtr5g106000 is located on
chromosome 5 (see Supplemental Data Set 1 online; Figure 2A).
3-Hydroxy-3-methylglutaryl CoA reductases (HMGRs) are en-
coded by amultigene family in plants, and five isoforms are found
in M. truncatula (Kevei et al., 2007). HMGR isoform 2 is repre-
sented by five copies located back-to-back on chromosome 5
(see Supplemental Data Set 1 online; Figure 2A). Four HMGR2b
copies are identical, including the intron sequences, whereas
HMGR2a has 98% nucleotide identity to these and a different
intron sequence.
HMGR isoforms 1 and 2 have 94% nucleotide sequence iden-
tity and are represented by the same probe sets, Mtr.10397.1.
S1_at, on the Affymetrix chip. All five isoforms were strongly
upregulated by MJ (5- to 30-fold at 24 h), whereas none of them
were induced by YE (see Supplemental Data Set 1 online).
HMGR isoforms 1 to 4, along with HMG-CoA synthase, are all
located on chromosome 5, whereas HMGR isoform 5 is located
on chromosome 8 (Figure 2A). HMGR isoforms 1 and 2 show the
most similar expression pattern to that of b-AS, the entry point
enzyme into the triterpene pathway (see Supplemental Figure 2
online).
A gene encodingmevalonate kinase (found on chromosome 7)
was upregulated by MJ but not YE (see Supplemental Data Set
1 online; Figures 1 and 2A). However, phosphomevalonate
Genomics of Saponin Biosynthesis 851
kinase (on chromosome 3) and mevalonate diphosphate decar-
boxylase, which together convert phosphomevalonate to IPP,
were not significantly upregulated. Microarray analysis also
failed to detect significant changes in transcripts encoding IPP
isomerase (chromosome7), which converts IPP into dimethylallyl
diphosphate.
The second phase of the terpenoid pathway involves con-
densation of allylic pyrophosphates with IPP to produce the
higher prenyl pyrophosphates (Alonso andCroteau, 1993), which
are converted to squalene for biosynythesis of triterpenes and
sterols (Bramley, 1997; Figure 1). Two prenyl transferases (FPS1,
Medtr2g032930, on chromosome 2; and SSU, Medtr5g100210,
on chromosome 5) were upregulated by MJ (see Supplemental
Data Set 1 online; Figure 2A). Only one squalene synthase gene,
Medtr4g097000 (chromosome 4), was found in the Medicago
genome, and this was induced 4.5-fold by MJ at 24 h after
elicitation (Figures 1 and 2A; see Supplemental Data Set 1 online).
Three squalene epoxidase (SE) genes most likely exist in Medi-
cago; however, only one gene, Medtr4g122000, has been se-
quenced so far. Two SE genes, including the previously reported
SE2 (Suzuki et al., 2002), were induced almost 2- to 3-fold at 24 h
after MJ treatment (see Supplemental Data Set 1 online).
Identification and Induction ofMedicago
2,3-Oxidosqualene Cyclases
The first committed step in the biosynthesis of triterpene
saponins and steroids involves the initial cyclization of 2,3-
oxidosqualene by 2,3-oxidosqualene cyclases into one of a
number of different potential products (Haralampidis et al.,
2002). There are two routes of cyclization of 2,3-oxidosqualene
to sapogenins, either via the chair-chair-chair or the chair-boat-
chair conformations (Vincken et al., 2007). Sterol cyclization
proceeds via the chair-boat-chair conformation (Haralampidis
et al., 2002; Vincken et al., 2007; Wang et al., 2008), catalyzed by
cycloartenol synthase (CAS) or lanosterol synthase (LS) (see
Figure 1. Changes in Transcript Levels of Genes Potentially Involved in Terpenoid Biosynthesis in M. truncatula Cell Cultures.
Green/red color-coded heat maps represent relative transcript levels of different gene family members determined with Affymetrix arrays; red,
upregulated; green, downregulated. After exposure to YE for 2 h (A); after exposure to MJ for 24 h (B). Data represent log2 scale ratios of transcript
levels in elicited compared with control cells. MapMan (Thimm et al., 2004) visualization software was used to depict transcript levels. HMG-CoA,
3-hydroxy-3-methylglutaryl CoA; MVA, mevalonic acid; MVAP, mevalonic acid 5-phosphate; PMVK, phosphomevalonate kinase; MVD, MVA
diphosphate decarboxylase; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl
diphosphate; SS, squalene synthase; 2,3-OSCs, 2,3-oxidosqualene cyclases; P450, Cytochrome P-450; GTs, glycosyltransferases.
852 The Plant Cell
Figure 2. Genetic Linkage and Coexpression of Potential Genes of Triterpene Metabolism in M. truncatula.
(A) Map positions of genes potentially involved in isoprenoid-triterpenoid pathways on chromosomes of M. truncatula.
(B) Microarray transcript level profiles of cluster of UGTs on chromosome 2.
(C) Microarray transcript level profiles of genes potentially involved in triterpene metabolism on chromosome 4.
Expression data were obtained from theM. truncatulaGene Expression Atlas database version 2 (MtGEAv2), which combines a large number of publicly
Genomics of Saponin Biosynthesis 853
Supplemental Figure 3 online). Initial triterpene cyclization prod-
ucts with the chair-chair-chair conformationmay be converted to
dammarene-like or pentacyclic triterpenoids, including lupeol,
a-amyrin, and b-amyrin (Haralampidis et al., 2002; Vincken et al.,
2007). These cyclization events are catalyzed by dammarenediol
synthase, lupeol synthase (LuS), a-amyrin synthase, or b-AS,
respectively (see Supplemental Figure 3 online).
Eight genes, showing unique expression patterns and simi-
larity to known 2,3-oxidosqualene cyclases, are present in
the Medicago genome (see Supplemental Data Set 2 online).
The expression of Medtr5g00886, represented by probe set
Mtr.38244.1.S1_at, is quite evenly distributed among the tissues,
likely reflecting a role in primary metabolism (see Supplemental
Figure 4 online). Phylogenetic analysis places this gene into the
CAS clade, with the closest homolog (92% amino acid identity)
being the CAS from P. sativum (Figure 3).
TC132384 (genomic sequence not available) was represented
by two probe sets, Mtr.38219.1.S1_at and Mtr.4710.1.S1_s_at,
showing similar expression patterns with the highest expression
level in nodules (shown for Mtr.38219.1.S1_ in Supplemental
Figure 4 online). TC132384 corresponds to the geneGB#Y15366
identified in an M. truncatula root nodule cDNA library (Gamas
et al., 1996) and annotated as encoding a CAS. However,
phylogenetic analysis revealed that this gene belongs to the
LuS clade, with highest homology (92%) to the LuS from Lotus
japonicus (Figure 3; see Supplemental Data Set 3 online). The
function of this gene, which was not induced by either YE or MJ
treatments, may be questionable in Medicago species where
lupeol derivatives have yet to be detected.
The probe set Mtr.9365.1.S1_at representing Medtr6g099000
showed expression specifically in roots and nodules (see Sup-
plemental Figure 4 online). Phylogenetic analysis placed this
gene in the LS clade (Figure 3). LS is conserved among the
eudicots; however, its function in plants is still unknown. This
gene was not induced in response to YE or MJ.
Oxidosqualene cyclase (Mtr.36453.1.S1_at) showed expres-
sion in leaves and petioles (see Supplemental Figure 4 online).
This gene is represented by a single EST, and its genomic
sequence is not yet available. It was not induced by YE or MJ.
Three genes, Medtr8g018540_50, Medtr8g018580_620,
and Medtr8g018630_60, are situated back-to-back on chro-
mosome 8. These genes are specifically expressed in flower
tissue and showed similar expression patterns (shown only for
Medtr8g018580_620 in Supplemental Figure 4 online). The
amino acid sequence of each of them showed from 81 to 85%
identity to that of amixed AS fromP. sativum (Morita et al., 2000).
Phylogenetic analysis revealed that these genes belong to the
a/b-amyrin cluster (Figure 3). None of these genes are expressed
in response to either YE or MJ.
Only one b-AS gene, represented by probe set Mtr.18630.1.
S1_at, was highly induced by MJ but not by YE (see Supple-
mental Data Set 1 online). This gene (GenBank accession num-
ber CAD23247) was functionally characterized previously by
expression in yeast and the recombinant enzyme shown to
convert 2,3-oxidosqualene to b-amyrin (Suzuki et al., 2002). It
exists as two copies in the M. truncatula genome (Suzuki et al.,
2002), although genomic sequence is currently available for only
one copy on chromosome 4 (Medtr4g163370).
TheM. truncatula b-AS gene clustered in the b-AS clade, with
the closest putative ortholog from P. sativum (Figure 3). Mining
the Medicago Gene Expression Atlas database (Benedito et al.,
2008) showed that b-ASwasmost highly expressed in seeds at a
late developmental stage and in roots (see Supplemental Figure
4 online).
The above studies define the biosynthetic potential of M.
truncatula regarding cyclization of oxidosqualene. We next
focused our attention on the identification of enzymes potentially
involved in the hydroxylations of the carbon skeletons and
transfer of sugar moieties to generate the structurally diverse
complement of Medicago saponins.
Selection of Candidate Cytochrome P450s
Cytochrome P-450 enzymes are membrane-associated hemo-
protein monooxygenases that are involved in a number of
biosynthetic and detoxification pathways in plants (Werck-
Reichhart et al., 2002). Two main classes of P450s, containing
10 clans and 62 families, have been identified in plants (Li et al.,
2007). Recently, 151 putative P450 genes from M. truncatula
were classified into nine clans and 44 families (Li et al., 2007). To
find potential P450 candidates involved in saponin biosynthesis,
we first searched the ongoing M. truncatula genome sequence
(http://gbrowse.jcvi.org/cgi-bin/gbrowse/medicago_imgag/)
and the probe sets of the AffymetrixMedicago chip (http://www.
affymetrix.com). We found 184 P450 genes with probe sets
present on the Affymetrix array and 37 probe sets for which
genomic sequences are not yet available (some of which may be
redundant; see Supplemental Data Set 4 online). Annotation of
P450swas based on a domain search of the InterPro andUniprot
databases. The functional prediction of these genes was based
on sequence similarity to previously characterized enzymes by
BLASTX search of the plant nonredundant database.
Since MJ triggers saponin accumulation in Medicago, we
first searched for MJ-inducible P450s;;10% of the P450 probe
sets were upregulated by MJ (see Supplemental Data Set 4
online). Mtr.8618.1.S1_at (genomic sequence not available) cor-
responds to TC100810, which shares 90% homology at the
amino acid level with b-amyrin and sophoradiol 24-hydroxylase
Figure 2. (continued).
available Medicago GeneChip microarrays (156 chips from 64 experiments). References with detailed descriptions of experiments presented in
MtGEAv2 are available on the website http://bioinfo.noble.org/gene-atlas/v2/. HMGcAS, 3-hydroxy-3-methylglutaryl CoA synthase; HMGcAR,
3-hydroxy-3-methylglutaryl CoA reductase; MVK, mevalonate kinase; PMVK, phosphomevalonate kinase; IPPI, isopentenyl pyrophosphate isomerase;
GGPS, geranylgeranyl pyrophosphate synthase; SSU, small subunit of GGPS; FPS1, farnesyl pyrophosphate synthase 1; SS, squalene synthase; UGT,
uridine diphosphate glycosyltransferase. Up- and down-pointing arrows represent forward or reverse orientations on a chromosome, respectively.
854 The Plant Cell
(CYP93E1) from Glycine max (GenBank accession number
BAE94181) (Shibuya et al., 2006), and was very strongly upregu-
lated by MJ (532-fold at 24 h). Several highly MJ-induced P450s,
such as Medtr4g032760, Medtr4g032910, Mtr.37299.1.S1_at,
and Mtr.37298.1.S1_at, showed similarity with the CYP72A
family. CYP72A1 from Catharanthus roseus was characterized
as a secologanin synthase involved in the biosynthesis of terpene
indole alkaloids (Irmler et al., 2000). Mtr.43018.1.S1_at, previ-
ously classified as CYP716A12 (GenBank accession number
ABC59076; Li et al., 2007), shares 46% amino acid homology
with CYP720B1 (GenBank accession number Q50EK6) from
loblolly pine (Pinus taeda) an enzyme that catalyzes oxidations of
multiple diterpene alcohol and aldehyde intermediates (Ro et al.,
2005). Overall, the above similarities to the few functionally
characterized P450s of terpenoid metabolism suggest that sev-
eral of the genes in Supplemental Data Set 4 online are good
candidates for involvement in triterpene hydroxylation.
Selection of Candidate UGTs
To date, only two Medicago UGTs with specificity for the
triterpene aglycones hederagenin, soyasapogenols B and E,
and medicagenic acid have been biochemically characterized
(Achnine et al., 2005), and the triterpene-specific UGT74M1
catalyzes C-28 glycosylation for formation of monodesmosides
in S. vaccaria (Meesapyodsuk et al., 2007). There are no reliable
methods to identify the substrate specificity of plant glycosyl-
transferases based on sequence similarity alone (Modolo et al.,
2007). One hundred sixty-three probe sets (based on domain
searching of genome and Affymetrix probe sets) were therefore
selected for correlation analysis with candidate P450s and b-AS;
;15% of the UGTs were highly MJ inducible (see Supplemental
Data Set 5 online). Most were annotated as (iso)flavonoid glyco-
syltransferases. The geneMedtr4g032770, corresponding to the
previously reported UGT73K1 with specificity for hederagenin
and soyasapogenols B and E (Achnine et al., 2005), was
upregulated 359-fold by MJ at 24 h after elicitation.
Cluster Analysis for Gene Function Prediction
Enzymes involved in the same function will be temporally and
spatially coexpressed. In some cases, such coexpression is
associated with the operation of metabolic channels (Jorgensen
et al., 2005). Based on this principle, we applied clustering
Figure 3. Phylogenetic Analysis of OSCs from M. truncatula and Other Plant Species.
GenBank accession numbers are provided on the tree leaves. Panax ginseng dammarenediol synthase represents an outgroup taxon. M. truncatula
OSCs are indicated by blue font. Branches of different enzyme classes are colored: green, a/b amyrin synthases; maroon, cycloartenol synthases;
sienna, lanosterol synthases; and black, lupeol synthases. A multiple alignment of the deduced amino acid sequences of OSCs and a phylogenetic tree
were constructed using the A la Carte mode (Muscle 3.7 for multiple alignment; Gblocks 0.91b for alignment refinement; MrBayes 3.1.2 for phylogeny
using maximum likelihood 6 number of substitution types, default substitution model, invariable + g rates variations, MCMC 10,000 generations;
TreeDyn 198.3 for Tree rending) of the Phylogeny.fr program (Dereeper et al., 2008). Posterior probabilities (marked by red font) are indicated near
nodes (MrBayes, 10,000 generations). The bar indicates the branch length that corresponds to 0.1 substitutions per position.
Genomics of Saponin Biosynthesis 855
analysis to find P450s andUGTswith themost similar expression
pattern to b-AS, the entry point enzyme into the triterpene
saponin pathway. For clustering analysis, we used expression
data obtained from the Medicago truncatula Gene Expression
Atlas database version 2 (MtGEAv2), which combines a large
number of publicly available Medicago GeneChip microarrays
(156 chips from 64 experiments; http://bioinfo.noble.org/gene-
atlas/v2/). Signal intensities were converted into log2 scale; data
for P450s and UGTs are provided in Supplemental Data Sets
4 and 5 online. Genes that did not show a detectable level of
expression were excluded from analysis to reduce noise. Three
hundred and fifteen probe sets, including b-AS, the P450s,
UGTs, and early pathway enzymes (HMGS, HMGRs, squalene
synthases, and SEs), were interrogated by hierarchical cluster
analysis based on Pearson’s correlation (with ranges from +1 to
21 where +1 is the highest correlation). Profiles with identical
shapes have maximum positive correlation. Perfectly mirrored
profiles have the maximum negative or inverse correlation.
Since, here, we are interested only in triterpene metabolism,
the cluster with similar profiles to b-AS is provided in this study
(Figure 4). This cluster includes nine UGTs, six P450s, b-AS, and
three HMGR probe sets, which exhibited correlation values of
0.956 or greater.
It has recently been reported that operon-like gene clusters
in Arabidopsis and oat are required for triterpene biosynthesis
and that such clusters assemble de novo under evolutionary
pressure (Field and Osbourn, 2008). To test this concept for
triterpene metabolism in M. truncatula, we interrogated the
ongoing M. truncatula genome sequence (http://www.tigr.org).
Five genes likely associated with triterpene production were
located on chromosome4, includingb-AS (Medtr4g163370), two
CYP72A61s (Medtr4g032760 and Medtr4g032910), UGT73K1
(Medtr4g032770), and UGT (Medtr4g130290; Figure 2A). Al-
though all these five genes are not tightly linked on chromosome
4, their expression profiles are very similar (Figure 2C) and
the two CYP72A61s, Medtr4g032760 and Medtr4g032910, and
UGT73K1 (Medtr4g032770) were closely linked. UGT73K1 is
known to glycosylate multiple sapogenins (Achnine et al., 2005),
and it is tempting to speculate that the linkedP450 introduces the
hydroxyl group that is subsequently glycosylated. We also found
that three UGTs, Medtr2g008360 (GT4), Medtr2g008370 (GT2),
and Medtr2g008380, are linked on chromosome 2 (Figure 2A).
The expression profiles of these three UGTs are very similar
(Figure 2B), and GT4 and Medtr2g008380 share 93% nucleotide
sequence identity. Two UGTs found on chromosome 7 (Figure
2A) also showed a similar expression pattern.
Functional Characterization of a Predicted Triterpene
UGT in Vitro
To confirm that coexpression analysis can correctly predict
genes involved in triterpene saponin biosynthesis, we selected
four currently uncharacterized UGT genes from the b-AS ex-
pression cluster described above. Although the significance of
Figure 4. The b-AS Cluster: Hierarchical Clustering Analysis of Gene Expression Patterns.
Transcript levels were measured in the different tissues (microarray data were obtained from Atlas database version 2, MtGEAv2, http://bioinfo.noble.
org/gene-atlas/v2/). The dendrogram represents hierarchical similarity in microarray-determined transcript profiles of genes potentially involved in
triterpene metabolism. The vertical axis of the dendrogram consists of the individual records, and the horizontal axis represents the clustering level. The
scale above the row dendrogram is the cluster slider. The numbers above the scale refer to the number of clusters at different positions in the dendrogram.
The numbers below the scale refer to the calculated similarity measures. The color scale above the cluster reflects the signal intensity converted to log2. The
part of the dendrogram shadowed in gray passed a cluster significance test, using Pvclust analysis (Suzuki and Shimodaira, 2006), with approximately
unbiased and bootstrap probability values of >95% (i.e., the hypothesis that “the cluster does not exist” is rejected; P value <0.05).
856 The Plant Cell
the clustering had a P value of >0.05 for some of these genes
(Figure 4), they were included because none was expressed in a
tissue or treatment in which b-AS was not expressed (the
significance level is reduced because some of the UGTs are
root-specific, whereas saponins, and thereforeb-AS expression,
are also found in the aerial parts in M. truncatula). We selected
UGT candidates over P450s because UGTs are easy to express
in E. coli, and it is not possible to predict UGT function based on
sequence similarity alone. The selection was mainly based on
availability of full-length sequences. The genomic sequence of
GT1 (GenBank accession number FJ477889) is available, locus
Medtr7g076740 on chromosome 7; GT2 (FJ477890) and GT4
(FJ477892) are from the cluster of three UGTs on chromosome 2
(Figure 2B); the genomic sequence of GT3 (FJ477891) is not yet
available, but the corresponding TC94916 represents a full-
length sequence. BLAST analysis revealed that all four UGTs
contain the conserved PSPG domain characteristic of the nu-
cleotide sugar binding site of small molecule UGTs. The four
selected UGTs have low amino acid sequence identity to each
other, except for GT2 and GT4, which shared 56% identity. GT1
shared 53% amino acid identity to an (iso)flavonoid UGT from
M. truncatula (ABI94026); GT2 and GT4 shared 47 and 43%
sequence identity, respectively, to a putative UDP-rhamnose,
rhamnosyltransferase from Fragaria x ananassa (AAU09445); and
GT3 shared 65% sequence identity to an isoflavonoid UGT from
G. echinata (BAC78438). These UGTs have subsequently been
assigned as UGT73P3 (GT1), UGT91H5 (GT2), UGT73F3 (GT3),
and UGT91H6 (GT4) by the UGT Nomenclature Committee
(Mackenzie et al., 1997).
To determine whether the selected UGTs encode functional
enzymes, their His-tagged fusion proteins were expressed in
E. coli Rosetta 2 (DE3) pLysS cells and affinity purified. An
SDS-PAGE gel of purified His-tagged GT3 and GT4 fusion pro-
teins is shown in Supplemental Figure 5 online. The glycosyltrans-
ferase activities of the recombinant proteinswere first tested using
UDP-glucose as the sugar donor and a range of potential acceptor
substrates, including hormones, steroids, triterpenoids, and fla-
vonoids (see Supplemental Figure 6 online). This selection was
based on the fact that cytokinins (some), gibberellins, abscisic
acid, cucurbitanes, brassinosteroids, and triterpenes are products
of the terpenoid pathway and that at least one UGT active with
triterpenes can also glycosylate flavonoids (Achnine et al., 2005).
GT3 (UGT73F3) showed activity with UDP-glucose as donor
and triterpene aglycones or the flavonol kaempferol as sugar
acceptors. The other threeUGTs did not show activity with any of
the tested acceptors using either UDP-glucose, UDP-galactose,
or UDP-glucuronic acid as donors. This could be because
expression in a prokaryotic system resulted in inactive enzymes,
the range of tested sugar donors and acceptors was not exten-
sive enough, or the UGTs function to add additional sugars to a
mono- or diglycosidic saponin.
Products from the reaction of UGT73F3with a crudemixture of
sapogenins were analyzed by HPLC–electrospray ionization–
mass spectrometry (ESI-MS). Figure 5A shows a selected base
peak chromatogram in which the sapogenin substrates are color
coded blue and their glucosylated derivatives are color coded
red. Seven major peaks were detected in the crude sapogenin
extract, three of which were identified as medicagenic acid (MS
502-H), bayogenin (MS 488-H), and hederagenin (MS 472-H).
Five compounds were glucosylated by UGT73F3, including the
above three (Figure 5). No products were observed with UDP-
galactose as sugar donor.
A radioactive assay using UDP-[U-14C] glucose was used to
determine the kinetic properties of purified recombinant
UGT73F3 with commercially available triterpene sapogenins
and kaempferol (Table 1). UGT73F3 was highly efficient with
hederagenin as sugar acceptor, exhibiting the highest Kcat/Km
ratio and turnover rate (Kcat value). The lowest Km was observed
for soyasapogenol A as substrate. However, the turnover of this
substrate was quite slow (Table 1). The substrate specificity of
UGT73F3 was approximately the same toward soyasapogenols
A and B (Table 1). We could not determine the Km value for
kaempferol since the saturation curve displayed a sigmoidal rate
substrate concentration relationship.
To determine the regiospecificity of UGT73F3, we performed
NMR analysis of the glucosylated product of hederagenin. The
combination of TOCSY, gCOSY, gHSQC, and gHMBC spectra
enabled us to determine all of the proton and carbon chemical
shifts of the hederagenin glucoside. The assignment of the peaks
is given in Supplemental Tables 1 and 2 online. The proton and
carbon chemical shifts of the anomeric position of the glucose
residue indicate that it is O-linked to C-28 of hederagenin in an
ester linkage (Figure 5B).
Genetic Loss-of-Function Analysis of UGT73F3
To determine whether UGT73F3 functions in triterpene saponin
biosynthesis in vivo, we performed loss-of-function genetic
analyses by screening pooled DNA from the M. truncatula Tnt1
retrotransposon insertion population (Tadege et al., 2008) with
primers specific for UGT73F3. Two mutant lines, NF8981 and
NF5746, were isolated. NF8981 was found to have the Tnt1
insertion at position 185 relative to the translation start site of
UGT73F3, whereas line NF5746 contained an insertion at posi-
tion 650 (see Supplemental Figure 7A online). Only two plants
germinated from seven seeds of line NF5746, and one of them
was confirmed to be heterozygous (see Supplemental Figure 7B
online). PCR analysis of genomic DNA of seven plants (R1
progeny) of line NF8981, using combinations of gene-specific
primers for UGT73F3 and the Tnt1 retrotransposon, revealed
that lines 6 and 7 were homozygous, whereas lines 1 and 3 were
heterozygous (see Supplemental Figure 7B online). RT-PCR
confirmed no expression of UGT73F3 in homozygous NF8981
lines, while expression levels in heterozygous lines were not
significantly different from thewild type (t test, P value >0.14) (see
Supplemental Figure 7C online). Flanking sequence tag analysis
of two homozygous plants of line NF8981 detected seven Tnt1
retrotransposon insertions in the genome of line 6 and 15
insertions in the genome of line 7 (see Supplemental Data Set 6
online). Five insertions were the same in both lines but did not
interrupt any known protein except UGT73F3. BLASTX analysis
against the nonredundant protein sequence database revealed
that in most cases the Tnt1 retrotransposon incorporated into
hypothetical or putative proteins of unknown function.
Unexpectedly, homozygous plants were retarded in
growth and never reached the size of normal plants, whereas
Genomics of Saponin Biosynthesis 857
Figure 5. HPLC-MS Analysis of the Products of GT3 Activity with a Crude Sapogenin Extract from Medicago Roots.
(A) Selected base peak chromatogram of the crude sapogenin extract (blue) and glucosylated products (red).
(B) Structure of hederagenin 28-O-b-D-glucopyranoside.
(C) to (H) Negative-ion HPLC-ESI-MS/MS of sapogenins and their glucosides produced by the action of UGT73F3 on a crude sapogenin extract
from M. truncatula: bayogenin glucoside (C), unidentified A glucoside (D), unidentified B glucoside (E), hederagenin glucoside (F), medicagenic
acid aglycone (G), and medicagenic acid glucoside (H). Explanation of masses for molecules indicated in square brackets: M, molecule; Glc, b-D-
glucopyranosyl; Hac, acetic acid; Na, sodium; and H, hydrogen.
858 The Plant Cell
heterozygous plants did not show anymorphological differences
compared with the wild type (see Supplemental Figure 7D
online). However, the homozygous plants could flower and
produce a few pods. The seeds did not show any visible
morphological changes but took an unusually long time to
germinate (at least 3 weeks). Root growth was severely affected
in homozygous lines; roots of these plants were very short and
less branched compared with the wild type.
Four plants of one homozygous line, NF8981-6, have survived
and show the same dwarf phenotype (Figure 6). Fifty seeds of
heterozygous line NF5746 have been screened and two homozy-
gous plants were detected; only one of them survived, and this R2
generation plant showed the same dwarf phenotype (Figure 6).
Saponin content was evaluated in root and leaf tissue of
8-week-old plants by HPLC-ESI-MS analysis. There were no
significant changes in leaf saponin levels between controls and
mutants. This was not surprising since the main site of UGT73F3
expression is roots, and the saponins in the aerial parts of
M. truncatula are primarily glucuronic acid conjugates (Huhman
et al., 2005; Kapusta et al., 2005b).
Roots from each of two individual plants (from four plants in
total) of line NF8981 were pooled into one sample to obtain
sufficient tissue for saponin analysis. Six different saponins have
been identified in M. truncatula root extracts according to pre-
viously published work (Huhman and Sumner, 2002; Huhman
et al., 2005; Kapusta et al., 2005a, 2005b), and mass data
for some are provided in Supplemental Figure 8 online. Levels
of Rha-Hex-Hex-Hex-hederagenin, Hex-Hex-Hex-bayogenin,
3-Glc-28-Glc-medicagenic acid, 3-Glc-Ara-28-Glc hederagenin,
and Hex-Hex-Hex-soyasapogenol E were significantly (P value
<0.05) reduced in mutant lines compared with controls, by
around threefold overall (Figures 7A and 7C). Only one saponin,
3-Glc-28-Ara-Rha-Xyl-medicagenic acid, was significantly in-
creased in the mutant lines. Reduction of 3-Glc-28-Glc-medica-
genic acid and 3-Glc-Ara-28-Glc hederagenin levels in knockout
lines is consistent with the in vitro regiospecificity of UGT73F3 for
C-28 and confirms in vivo the involvement of UGT73F3 in
glucosylation of multiple sapogenins in Medicago. Although
UGT73F3 was active with kaempferol in vitro, neither kaempferol
nor its conjugates were detected in M. truncatula roots.
In 3-Glc-28-Ara-Rha-Xyl-medicagenic acid, the 28-position is
substituted with arabinose, and glucosylation occurs only at the
C-3 hydroxyl position. The large (10-fold) increase of this com-
pound in UGT73F3 knockout lines suggests that the UDP-
glucose pool is being diverted toward increased formation of
non-C-28-glucosylated sapogenins. Levels of the isoflavone
formononetin and its conjugates were also significantly (P value
<0.05) increased inUGT73F3 knockout lines (Figures 7A and 7D).
DISCUSSION
Chromosomal Location and Coexpression of Saponin
Biosynthetic Genes inMedicago
The early enzymes common to multiple branches of terpenoid
metabolism are often encoded bymultigene families in plants. To
Table 1. Kinetic Parameters for UGT73F3
Substrate Vmax (mmol/min) Km (mM) Kcat (s�1)
Kcat/Km
(s�1 M�1)
Hederagenin 20.4 6 0.98 220.3 0.190 862.5
Soyasapogenol B 1.8 6 0.23 92.7 0.017 183.4
Soyasapogenol A 0.8 6 0.03 46.7 0.008 171.3
Errors represent SD from triplicate measurements.
Figure 6. Growth Phenotype of UGT73F3 Mutants.
Eight-week-old UGT73F3 tnt1 homozygous mutant lines NF5746 and NF8981 (R2 generation plants) are stunted in growth compared with wild-typeM.
truncatula (R108 var) plants.
[See online article for color version of this figure.]
Genomics of Saponin Biosynthesis 859
Figure 7. Targeted Metabolic Profiles of Roots of Wild-Type and Mutant M. truncatula.
(A) and (B) Full scan (A) and selected negative-ion HPLC-ESI-MS chromatograms (B) at m/z 269 of M. truncatula root extract of control line R108-1
(black line) and UGT73F3 tnt1 knockdown line NF8981-1 (gray dashed line). F, formononetin; FG, formononetin 7-O-b-D-glucoside (ononin); FGM,
formononetin 7-O-b-D-glucoside-6”-O-malonate; MG, medicarpin 3-O-b-D-glucoside; MGM, medicarpin 3-O-b-D-glucoside-malonate; RHHHHed,
Rha-Hex-Hex-Hex-hederagenin; HHHBay, Hex-Hex-Hex-Bayogenin; 3G28GMed, 3-Glc-28-Glc-medicagenic acid; 3G28ARXMed, 3-Glc-28-Ara-Rha-
Xyl-medicagenic acid; 3GA28GHed, 3-Glc-Ara-28-Glc hederagenin; and HHHSoyE, Hex-Hex-Hex-Soyasapogenol E. Names of saponins (in [A]) are
represented by black font and isoflavonoids by gray font. The inset in (B) shows the positive ion mass spectrum of the MGM peak.
(C) and (D) Relative content of saponins and isoflavonoids, respectively, based on peak areas in roots of control and UGT73F3 tnt1 mutant lines of M.
truncatula. The controls represent three independent plants ofM. truncatula R108. Root tissues from each of two individual plants of the R2 generation
UGT73F3 tnt1 mutant line NF8981 were pooled into two samples, NF8981-1 and NF8981-2. The pooling was necessary due to the limited amount of
tissue from the dwarf mutant lines. Only one homozygous plant (R2 generation) was available for line NF5746 (no pooling for this line). Error bars indicate
SE from three biological replicates. All identified metabolites accumulated significantly differently in controls compared with mutant plants with a P value
of <0.05. Identification of saponins and isoflavonoids was based on previously published work (Huhman and Sumner, 2002; Huhman et al., 2005;
Kapusta et al., 2005a, 2005b; Farag et al., 2007). Explanations of masses for saponins are provided in Supplemental Figure 8 online.
860 The Plant Cell
assess those family members most likely associated with triter-
pene saponin biosynthesis in Medicago, we compared both
chromosomal localization and gene expression pattern for all
possible candidates.
HMGR is often regarded as the key regulatory gene in terpe-
noid metabolism. Five HMGR genes are present in the
M. truncatula genome, and the appearance of four identical
HMGR2b genes could be due to recent duplication events. The
Arabidopsis genome contains only two HMGR genes. The ex-
pansion of HMGR genes in the M. truncatula genome might be
related to the expansion of triterpene metabolism in this species.
Interestingly, several of the genes encoding the early steps in
terpene biosynthesis (thiolase, HMG-CoA synthase, most of the
HMGRs, and some IPP synthases) are found on chromosome 5.
Medicago HMGR1 has been shown to be critical for nodulation
(Kevei et al., 2007); since both isoforms 1 and 2 are tightly
coexpressed with b-AS, either might be involved in triterpene
biosynthesis, although functions for triterpenes in nodulation
have not been demonstrated.
A preliminary conclusion from our analyses of both the early
and (predicted) late genes of triterpene saponin biosynthesis is
that some of the genes might be clustered as a result of gene
duplication in M. truncatula but that, overall, these genes are not
assembled into operon-like clusters, as has been reported for
genes involved in some branches of triterpene biosynthesis in
oat and Arabidopsis (Qi et al., 2004; Field and Osbourn, 2008).
Chromosome 4 is the most likely site for the genes involved
specifically in triterpene biosynthesis in Medicago since, apart
from the presence of several of the early enzyme genes on
chromosome 5, most of the critical enzymes are found there.
These genes are not tightly linked; however, the maintenance of
their location on the same chromosomeduring evolution suggests
that theremaybesomebenefit fromhaving thesegenes physically
associated. It is possible that regulatory effects can operate over
relatively long distances on this region of chromosome 4.
Medicagenic acid conjugates are major constituents in the
aerial parts ofM. truncatula, whereas soyasapogenol conjugates
are major constituents in roots (Huhman et al., 2005; Kapusta
et al., 2005a). However, it is likely that a single b-AS is respon-
sible for production of most triterpene saponins in this species. A
truncated copy of a gene with high similarity to b-AS was found
on chromosome 8, next to three mixed ASs (see Supplemental
Data Set 2 online; Figure 2A); this may explain the two b-AS
copies observed on DNA gel blot analysis (Suzuki et al., 2002).
Our analysis of potential OSC genes predicts genes involved
in formation of triterpene classes yet to be discovered in
Medicago. Compounds with the a-amyrin skeleton have not
yet been reported in Medicago species. Further studies specif-
ically targetinga-amyrin–derived saponins inMedicago are clearly
warranted, since >90 different triterpene skeletal types can theo-
retically be generated by cyclization of 2,3-oxidosqualene (Morita
et al., 2000).
Determination of Candidate P450s and UGTs for Triterpene
Saponin Biosynthesis
Clustering analysis of transcript and metabolite profiles is be-
coming a powerful technique for identifying candidate genes in
complex plant secondary metabolic pathways (Yonekura-
Sakakibara et al., 2007, 2008; Saito et al., 2008; Shulaev et al.,
2008). In this study, the transcript profiles of a large number of
P450 and UGT genes clustered tightly with b-AS in regards
to both tissue-specific and elicitor-inducible expression. One
of these genes, encoding the glucosyltransfrease UGT73K1
with specificity for hederagenin and soyasapogenols B and E
(Achnine et al., 2005), had already been identified as being
involved in triterpene saponin biosynthesis, and TC100810
shares 90% amino acid identity with CYP93E1, a b-amyrin and
sophoradiol 24-hydroxylase from soybean (Shibuya et al., 2006).
These observations, along with the subsequent identification of
UGT73F3 as a triterpene UGT, validate the clustering approach
in Medicago and suggest that more of the UGT and P450 genes
listed in Figures 2 and 4 and Supplemental Table 1 online are
strong candidates for involvement in the saponin pathway. Our
work therefore provides the basis for future studies to define
genetically the roles of P450s and UGTs in triterpene saponin
biosynthesis in Medicago.
UGT73F3 Is a Saponin Glycosyltransferase
Five different triterpene aglycones, medicagenic acid, bayoge-
nin, hederagenin, and soyasapogenols B and E, have been
determined as base skeletons for >30 M. truncatula saponins
described previously (Huhman and Sumner, 2002; Suzuki et al.,
2002; Huhman et al., 2005; Kapusta et al., 2005a, 2005b).
UGT73F3 showed activity with at least four of these compounds
in vitro and also glucosylated the flavonol kaempferol. We would
not expect kampferol to be a natural substrate for UGT73F3 in
vivo since flavonoid glucoside levels were not increased in
response to MJ treatment of Medicago cell cultures (Farag
et al., 2008), and kampferol was not detected in roots, the main
site of UGT73F3 expression. Similarly, kaempferol is a good
substrate for Medicago UGT71G1 in vitro (Shao et al., 2005),
although this enzyme is also active with triterpene sapogenins
(Achnine et al., 2005).
Typically, glucosyltransferases exhibit substrate regiospeci-
ficity rather than absolute specificity for a particular compound in
vitro. For example, UGT85B1 from Sorghum bicolor showed a
broad activity spectrum in vitro with different families of accep-
tors (including cyanohydrins, terpenoids, phenolics, and hexanol
derivatives) that was influenced by the stereochemistry and/or
interactive chemistry of the substituents on the hydroxyl-bearing
carbon atom (Hansen et al., 2003). However, this may not reflect
the in vivo situation. For example, the glucosyltransferase
UGT78G1, which showed a strong preference for isoflavonoid
substrates in vitro, appears to function as an anthocyanin gly-
cosyltransferase in Medicago in vivo (Modolo et al., 2007; Peel
et al., 2009). Another example is TOGT1 from N. tabacum, which
showed activity with salicylic acid in vitro but not in vivo (Chong
et al., 2002).
M. truncatula and alfalfa sapogenins are glycosylated at the
C-3 and C-28 positions (Huhman and Sumner, 2002; Huhman
et al., 2005; Kapusta et al., 2005a, 2005b). Medicagenic acid and
bayogenin, like hederagenin, have carboxyl groups at the C-28
position (see Supplemental Figure 5 online), whichmay therefore
be glucose esterified through the action of UGT73F3. NMR
Genomics of Saponin Biosynthesis 861
analysis indicated that the glucoside produced by the action of
UGT73F3 with hederagenin as sugar acceptor was the C-28
ester. It is therefore likely that medicagenic acid and bayogenin
are also glycosylated at the 28 position by UGT73F3. Soyasa-
pogenols A, B, and E have no hydroxyl group at C-28; therefore,
only the hydroxyl at C-3 is available for glycosylation of these
compounds. Since UGT73F3 can glycosylate soyasapogenols A
and B, albeit relatively weakly, the enzyme is not strictly regio-
specific for triterpene glycosylation.
Phenotypic and Biochemical Effects of Loss of
UGT73F3 Function
The reduction in levels of C-28 glycosylated triterpenes in M.
trunctula lines harboring a retrotransposon insertion inUGT73F3
is strong evidence in support of a function for this enzyme in
saponin glycosylation in vivo. However, other characteristics of
these mutant lines require explanation. For example, isoflavone
glucosides are among the major secondary metabolites in
Medicago roots (Farag et al., 2007), and total isoflavone content
was ;2 times higher in UGT73F3 knockout lines than in cor-
responding controls. There are two possible interpretations for
this observation. First, blocking saponin glycosylation might
increase the endogenous pool of the sugar donor UDP-glucose,
thus leading to preferential synthesis of other glycosides. Alter-
natively, accumulation of nonglycosylated sapogenins might be
toxic, and the increased isoflavone accumulation might be a
nonspecific response to toxic stress (von Rad et al., 2001;
Bowles et al., 2005, 2006; Mylona et al., 2008).
The most striking phenotype of the UGT73F3 knockout lines is
the strong decrease in plant growth. Although our data do not
conclusively prove that this is a direct result of loss of function
of UGT73F3, the importance of glycosylation in cell division,
growth, and development in animals and plants has been sup-
ported by many research reports (Bowles et al., 2005, 2006),
some of which emphasize protective functions of glycosylation
against toxic compounds, including saponins. Thus, the sad3
and sad4 mutants of oat, deficient in a UGT, accumulate the
saponinmonodeglucosyl avenacinA-1,whichdisruptsmembrane
trafficking and causes degeneration of the epidermis, with con-
sequential effects on root hair formation (Mylona et al.,
2008). Loss-of-functionmutations in anArabidopsisUGT required
for glucosinolate biosynthesis gave a leaf chlorosis phenotype that
has been ascribed to the accumulation of toxic levels of thiohy-
droximate, the substrate for this enzyme (Grubb et al., 2004). A
root-expressed pea UDP-glycosyltransferase, UGT1, that glyco-
sylates flavonoids has been shown to be essential for plant
development, possibly via regulation of the cell cycle (Woo et al.,
1999).
In this work, we predicted at least nine UGTs, including the
previously characterized UGT73K1 (Achnine et al., 2005), that
might be involved in triterpene saponin biosynthesis. Assuming
that the growth phenotype is indeed the result of loss of function
of UGT73F3, it is hard to understand how dysfunction of only one
UGT will cause such severe growth phenotypes. Do the various
triterpene UGTs perform specific functions in glycosylation of
only one or at most a few compounds in vivo, or do they have
broader specificity? If the latter, why does redundancy not
protect the plant from adverse effects of downregulation of a
single enzyme? Either the 28-glycosylation of saponins is espe-
cially critical for biological activity, or perhaps UGT73F3 has yet
to be discovered functions beyond the triterpene pathway.
These questions can only be answered when the remaining
enzymes of triterpene substitution have been characterized. This
work sets the stage for this endeavor.
METHODS
Plant Material
Details of the initiation and elicitation of Medicago truncatula Gaerth
‘Jemalong’ (line A17) cell suspension cultures have been given previously
(Broeckling et al., 2005; Suzuki et al., 2005; Naoumkina et al., 2007; Farag
et al., 2008).
M. truncatula R108 tnt1 mutant lines were grown in 6.5-inch-diameter
pots containing Professional blend soil (Sun Gro Horticulture) at a
temperature of 208C/198C (day/night), 16 h/8 h light/dark regime, and
40% relative humidity. Plants were fertilized at the time ofwatering using a
commercial fertilizer mix [Peters Professional 20-10-20 (N-P-K) General
Purpose; The Scotts Company].
Chemicals and Biological Materials
UDP-[U-14C] glucose (300 mCi/mmol) was purchased from American
Radiolabeled Chemicals. Hederagenin and soyasapogenols A and B
were from Chromadex. Auxins (4-chlorophenoxyacetic acid, 2,4-D, and
indole-3-acetic acid), cytokinins (kinetin, 2-isopentenyl adenine, and
zeatin), gibberellic acid, abscisic acid, campesterol, and kaempferol
were from Sigma-Aldrich. Cucurbitacin D was from Extrasynthese (Z.I.
Lyon Nord). Medicarpin was extracted and purified from alfalfa roots as
described previously (Modolo et al., 2007). Sapogenin extracts for
profiling were obtained from M. truncatula (Jemalong, cv A17) and
Medicago sativa (cv Radius, Kleszczewska, and Apollo) roots using a
solid phase extraction technique described previously (Huhman and
Sumner, 2002).
Saponins for profiling were obtained from M. truncatula R108 tnt1
mutant lines by extraction of freeze-dried ground shoots tissue with 80%
methanol.
HPLC-ESI-MS Analysis
An Agilent 1100 series II HPLC system (Hewlett-Packard) equipped
with a photodiode array detector was coupled to a Bruker Esquire ion-
trap mass spectrometer via an ESI source. UV spectra were obtained
by scanning from 200 to 600 nm. HPLC separation used a reverse-
phase, C18, 5-mm, 4.6 3 250-mm column (J.T. Baker) eluted with
0.1% aqueous acetic acid (eluent A) and acetonitrile (eluent B) with
a linear gradient of 5 to 90% B (v/v) over 90 min. The flow rate was
0.8 mL min21, and the temperature of the column was maintained at
288C. Negative-ion ESI mass spectra were acquired. Nebulization
was aided with a coaxial nitrogen sheath gas provided at a pressure of
60 p.s.i. Desolvation was assisted using a countercurrent nitrogen
flow set at a pressure of 12 p.s.i. and a capillary temperature of 3008C.
Mass spectra were recorded over the range 50 to 2200 m/z. The
Bruker ion-trap mass spectrometer was operated using an ion current
control of ;10,000 with a maximum acquire time of 100 ms. Tandem
mass spectra were obtained in manual mode for targeted masses
using an isolation width of 2.0, fragmentation amplitude of 2.2, and
threshold set at 6000. HPLC-MS data files were analyzed using Bruker
Daltonics esquireLC.
862 The Plant Cell
DNAMicroarray Analysis
RNA samples for analysis using the Affymetrix GeneChip Medicago
Genome Array were prepared from cells exposed to YE or MJ for 2 or
24 h, along with the corresponding nonelicited controls. Two biological
replicates, with analytical duplicates, were used for minimal statistical
treatment, and mean values for each treatment were divided by the
corresponding control baseline values. Full details of the experimental
procedures have been presented elsewhere (Naoumkina et al., 2007).
Differentially expressed genes in treatment/control experiments
were selected using associative analysis as described (Dozmorov and
Centola, 2003). Type I family-wise error rate was reduced using a
Bonferroni-corrected P value threshold of 0.05/N, where N represents
the number of probe sets present on the chip. The gene selections were
further confirmed by significance analysis of microarrays (Tusher et al.,
2001). The false discovery rate was monitored and controlled by calcu-
lating the Q value (false discovery rate) using extraction of differential
gene expression (http://www.biostat.washington.edu/software/jstorey/
edge/) (Storey and Tibshirani, 2003; Leek et al., 2006). The complete
Affymetrix data set is publicly available at ArrayExpress (http://www.ebi.
ac.uk/arrayexpress; ID = E-MEXP-1092).
Cluster Analysis
Expression data for selected genes were obtained from the Medicago
truncatula Gene Expression Atlas database version 2 (MtGEAv2), which
combine a large number of publicly available Medicago GeneChip
microarrays (156 chips from 64 experiments). References with a detailed
description of the experiments presented inMtGEAv2 are available on the
website http://bioinfo.noble.org/gene-atlas/v2/. Hierarchical clustering
analysis was performed with Spotfire DecisionSite 8.1. Data were trans-
formed to log2 and clustered using Pearson correlation analysis (Zar,
1999). Statistical analysis of uncertainty in hierarchical clustering was
performed with Pvclust software (Suzuki and Shimodaira, 2006).
RNA Isolation, Cloning, and Expression of UGTs
Total RNA was isolated from 0.5 g of frozen, ground M. truncatula
suspension cells using 5 mL of Tri-Reagent (Molecular Research Center)
following the manufacturer’s protocol. One microgram of total RNA was
used in a first-strand synthesis using SuperScript III reverse transcriptase
(Invitrogen) in a 20-mL reaction with oligo(dT) primers according to the
manufacturer’s protocol. A 2-mL aliquot of the first-strand reaction was
then PCR amplified for 30 cycles at 608C annealing temperature using
KOD Hot Start DNA polymerase (EMD Chemicals) according to the
manufacturer’s protocol.
Candidate UGTs were cloned into Gateway pENTR cassettes (Invitro-
gen). The inserts were transferred into the destination vector pDEST17 for
expression in Escherichia coli using the LR recombination reaction.
Primers for Gateway cloning were as follows: GT1, forward 59-CAC-
CATGGAGTCTCAACAATCCCATAAC-39, reverse 59-CTAATCTGCTTT-
CACACCAAGTGCCTTA-39; GT2, forward 59-CACCATGGATAACAA-
GAAAAACAAACCTCTTCA-39, reverse 59-CTATGAATTGTGATTTTGAA-
GTGAAGAAATGAAGT-39; GT3, forward 59-CACCATGGAAGGTGTTGA-
AGTTGAACAA-39, reverse 59-TTAATCATCCAGCTTGAGGTCTCTCA-
ATCT-39; GT4, forward 59-CACCATGGGTTCTACTGTTAATGAAGA-
AGA-39, reverse 59-TTAGTTGTTGGAATTGGAAGGAACCCTATAC-39.
E. coli Rosetta 2 (DE3) pLysS cells (Novagen) harboring the expression
construct were grown to an OD600 of 0.4 to 0.5, and expression was
initiated by addition of isopropyl 1-thio-b-D-galactopyranoside to a final
concentration of 0.2mM,with further incubationwith shaking overnight at
168C. The recombinant proteins were purified using the MagneHis
Protein Purification System according to the manufacturer’s protocol
(Promega).
Screening theM. truncatula Tnt1 Retrotransposon Insertion
Population for Identification of UGT73F3 Loss-of-FunctionMutants
The M. truncatula R108 Tnt1 population (Tadege et al., 2008) was
screened for insertions in the UGT73F3 sequence using the following
pairs of primers: GT3F1 forward, 59-ATGGAAGGTGTTGAAGTTGAACA-
ACC-39; GT3R1 reverse, 59-TTAATCATCCAGCTTGAGGTCTCTCA-39;
GT3F2 forward, 59-TATGTTTGCATCCCGTGGCCAGCAAG-39; and GT3R2
reverse, 59-CTCTCGATCTTTTAAGTTCGTCAATC-39. The line NF8981
was found to have the Tnt1 insertion at position 185 relative to the
translation start site of UGT73F3.
Ten seeds of NF8981were scarifiedwith concentrated sulfuric acid and
germinated for 5 d on moist sterile filter paper. Only seven seeds
germinated, and they were then planted in soil and screened by PCR
for identification of homozygous lines using the gene-specific primers
GT3F1-GT3R1 (shown above). To confirm the Tnt1 insertion, plants were
screened by PCR using one gene-specific primer, GT3R1, and one
retrotransposon-specific primer, Tnt1R, 59- CAGTGAACGAGCAGAA-
CCTGTG-39.
RT-PCR
RT-PCR was performed using a Quantum RNA 18S internal standard kit
(Ambion) according to the manufacturer’s protocol. RNA was isolated
from leaf tissue as described above. GT3F1 and GT3R1 primers (se-
quences shown above) were used to determine UGT73F3 transcript
levels in Tnt1 mutant lines. Actin (reference gene) was amplified by the
following: actin forward, 59-GGCTGGATTTGCTGGAGATGATGC-39; and
actin reverse, 59-CAATTTCTCGCTCTGCTGAGGTGG-39. Each RT-PCR
reaction was repeated three times independently. PCR products were
separated in a 1% agarose gel and stained with Syber Green (Invitrogen).
The fluorescence signal was captured using a UVP Bioimaging system.
Analysis of signal intensity of products was performed with Image Quant
TL software (Amersham Biosciences). Transcript abundance was deter-
mined by ratio to actin.
Enzyme Assays
Enzyme reactions were performed with 5 mg of enzyme in a total volume
of 50mL containing 50mMTris-HCl, pH 9.5, for GT3 (optimum) and pH7.0
for GT1, GT2, and GT4, 5.0 mM UDP-glucose, UDP-galactose, or UDP-
glucuronic acid, and 250mMacceptor substrate or 2 mg crude sapogenin
extract at 308C for 30 min. Samples were extracted with 250 mL of ethyl
acetate, and 225-mL aliquots were taken to dryness using a rotary
evaporator, diluted in 50 mL of methanol, and products analyzed by
HPLC-ESI-MS as described above.
For kinetic analysis of UGT73F3 (using three analytical replicates), 5 mg
of purified enzyme was added to reaction mixtures (50 mL final volume)
containing 50 mM Tris-HCl, pH 9.5, 1.7 mMUDP-[U-14C]-glucose (0.3 Ci/
mmol), 250 mM UDP-glucose (unlabeled), and 0 to 250 mM acceptor
substrate. Reactions were incubated for 30 min at 308C. Samples were
extracted with 250 mL of ethyl acetate, and 200 mL was taken for liquid
scintillation counting (Beckman LS6500). Data were analyzed using
Hyper32 software (http://www.liv.ac.uk/~jse/software.html).
NMR Spectroscopy of Hederagenin Glucoside
Hederagenin glucoside was generated by enzymatic reaction with 10 mg
of UGT73F3 protein in a total volume 100 mL containing 50 mM Tris-HCl,
pH 9.5, 5 mM UDP-glucose, and 220 mM hederagenin overnight at 308C.
The product was extracted with 3 volumes of ethyl acetate, dried under
nitrogen, and diluted in 8% methanol. Hederagenin glucoside was
purified using C18 SPE cartridges (Waters). Themobile phases consisted
of eluent A (0.1% aqueous acetic acid) and eluent B (acetonitrile). The
Genomics of Saponin Biosynthesis 863
SPE cartridges were equilibrated with three column volumes of 5% B
(v/v). The sample (half column volume) was loaded to the SPE cartridge,
which was washed with one column volume of 35% B (v/v). Hederagenin
glucoside was eluted with one column volume of 45% B (v/v) and dried
under a stream of nitrogen. Product (1.3 mg) was collected, deuterium
exchanged by lyophilization from D2O, and dissolved in 0.3 mL
pyridine-d5. One- and two-dimensional NMR spectra were acquired on
a Varian Inova-800 MHz spectrometer at 298K (258C). Proton chemical
shifts were measured relative to the most upfield pyridine-d5 singlet (dH =
7.22 and dC = 123.87 ppm).
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession numbers FJ477889 (UGT73P3), FJ477890
(UGT91H5), FJ477891 (UGT73F3), and FJ477892 (UGT91H6). Microarray
data are available at ArrayExpresss (http://www.ebi.ac.uk/arrayexpress,
ID = E-MEXP-1092).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Functional Categories of Genes That Are
Up- or Downregulated in Response to YE or MJ in M. truncatula
Suspension Cells.
Supplemental Figure 2. M. truncatula b-AS (Mtr.18630.1.S1_at) and
HMGR1-2 (Mtr.1039.1.S1_at) Expression Profiles (Atlas/v2).
Supplemental Figure 3. Cyclization of 2,3-Oxidosqualene to Sterols
and Triterpene Saponins.
Supplemental Figure 4. Microarray Analysis of the Tissue-Specific
Expression of Medicago 2,3-Oxidosqualene Cyclases.
Supplemental Figure 5. SDS-PAGE Gel of Purified Histidine-Tagged
Fusion Proteins Encoded by GT3 (UGT73F3) and GT4 (UGT91H6)
Genes.
Supplemental Figure 6. Structures of Substrates Tested with Re-
combinant Medicago UGT73F3 Expressed in E. coli.
Supplemental Figure 7. Growth Phenotype and PCR/RT-PCR Anal-
ysis of Mutants Harboring a Transposon Insertion in UGT73F3.
Supplemental Figure 8. Negative-Ion HPLC/ESI/MS/MS of Saponins
Extracted from Roots of M. truncatula Lines Harboring a Transposon
Insertion in UGT73F3.
Supplemental Table 1. NMR Chemical Shift Data for the Carbohy-
drate Portion of the Hederagenin Glycoside.
Supplemental Table 2. NMR Chemical Shift Data for the Aglycone
Portion of the Hederagenin Glycoside.
Supplemental Data Set 1. Chromosomal Positions of M. truncatula
Genes Involved in Terpenoid Metabolism.
Supplemental Data Set 2. 2,3-Oxidosqualene Cyclase Gene Names
and Probe Sets Available on the Medicago Affymetrix Chip.
Supplemental Data Set 3. Amino Acid Sequences of OSCs Used for
Phylogenetic Analysis.
Supplemental Data Set 4. Microarray Analysis of M. truncatula
P450s.
Supplemental Data Set 5. Microarray Analysis of M. truncatula
UGTs.
Supplemental Data Set 6. Flanking Sequence Tags in the Genomes
of Two Homozygous Plants (6 and 7) of Line NF8981.
ACKNOWLEDGMENTS
We thank Lahoucine Achnine (BASF Plant Sciences, Research Triangle
Park, NC) and Xiaoqiang Wang (Noble Foundation) for critical reading of
the manuscript and Parastoo Azadi (University of Georgia at Athens) for
NMR analysis. This work was supported by the National Science
Foundation Plant Genome Program Research Award DBI-0109732
and by the Samuel Roberts Noble Foundation. Any opinions, findings,
and conclusions or recommendations expressed in this material are
those of the authors and do not necessarily reflect the views of the
National Science Foundation. NMR analysis of hederagenin glucoside
was supported in part by the Department of Energy–funded (DE-FG09-
93R-20097) Center for Plant and Microbial Complex Carbohydrates at
the University of Georgia.
Received December 4, 2009; revised February 24, 2010; acceptedMarch
9, 2010; published March 26, 2010.
REFERENCES
Abe, I., Rohmer, M., and Prestwich, G.D. (1993). Enzymatic cyclization
of squalene and oxidosqualene to sterols and triterpenes. Chem. Rev.
93: 2189–2206.
Achnine, L., Huhman, D.V., Farag, M.A., Sumner, L.W., Blount, J.W.,
and Dixon, R.A. (2005). Genomics-based selection and functional
characterization of triterpene glycosyltransferases from the model
legume Medicago truncatula. Plant J. 41: 875–887.
Alonso, W.R., and Croteau, R. (1993). Prenyltransferases and cy-
clases. In Methods in Plant Biochemistry. Enzymes of Secondary
Metabolism, P.M. Dey and J.B. Harborne, eds (London: Academic
Press), pp. 239–260.
Behboudi, S., Morein, B., and Villacres Eriksson, M.C. (1999). Quillaja
saponin formulations that stimulate proinflammatory cytokines elicit
a potent acquired cell-mediated immunity. Scand. J. Immunol. 50:
371–377.
Benedito, V.A., et al. (2008). A gene expression atlas of the model
legume Medicago truncatula. Plant J. 55: 504–513.
Bowles, D., Isayenkova, J., Lim, E.K., and Poppenberger, B. (2005).
Glycosyltransferases: managers of small molecules. Curr. Opin. Plant
Biol. 8: 254–263.
Bowles, D., Lim, E.K., Poppenberger, B., and Vaistij, F.E. (2006).
Glycosyltransferases of lipophilic small molecules. Annu. Rev. Plant
Biol. 57: 567–597.
Bramley, P.M. (1997). Isoprenoid metabolism. In Plant Biochemistry,
P.M. Dey and J.B. Harborne, eds (London: Academic Press), pp.
417–434.
Broeckling, C.D., Huhman, D.V., Farag, M., Smith, J.T., May, G.D.,
Mendes, P., Dixon, R.A., and Sumner, L.W. (2005). Metabolic
profiling of Medicago truncatula cell cultures reveals effects of biotic
and abiotic elicitors on primary metabolism. J. Exp. Bot. 56: 323–336.
Chen, J.C., Chiu, M.H., Nie, R.L., Cordell, G.A., and Qiu, S.X. (2005).
Cucurbitacins and cucurbitane glycosides: structures and biological
activities. Nat. Prod. Rep. 22: 386–399.
Chong, J., Baltz, R., Schmitt, C., Beffa, R., Fritig, B., and Saindrenan,
P. (2002). Downregulation of a pathogen-responsive tobacco UDP-
Glc:phenylpropanoid glucosyltransferase reduces scopoletin gluco-
side accumulation, enhances oxidative stress, and weakens virus
resistance. Plant Cell 14: 1093–1107.
Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet,
F., Dufayard, J.F., Guindon, S., Lefort, V., Lescot, M., Claverie,
J.M., and Gascuel, O. (2008). Phylogeny.fr: Robust phylogenetic
analysis for the non-specialist. Nucleic Acids Res. 36: W465–469.
864 The Plant Cell
Dixon, R.A., and Sumner, L.W. (2003). Legume natural products.
Understanding and manipulating complex pathways for human and
animal health. Plant Physiol. 131: 878–885.
Dozmorov, I., and Centola, M. (2003). An associative analysis of gene
expression array data. Bioinformatics 19: 204–211.
Farag, M.A., Huhman, D.V., Dixon, R.A., and Sumner, L.W. (2008).
Metabolomics reveals novel pathways and differential mechanistic
and elicitor-specific responses in phenylpropanoid and isoflavonoid
biosynthesis in Medicago truncatula cell cultures. Plant Physiol. 146:
387–402.
Farag, M.A., Huhman, D.V., Lei, Z., and Sumner, L.W. (2007). Met-
abolic profiling and systematic identification of flavonoids and iso-
flavonoids in roots and cell suspension cultures of Medicago
truncatula using HPLC-UV-ESI-MS and GC-MS. Phytochemistry 68:
342–354.
Field, B., and Osbourn, A.E. (2008). Metabolic diversification - Inde-
pendent assembly of operon-like gene clusters in different plants.
Science 320: 543–547.
Frey, M., Chomet, P., Glawischnig, E., Stettner, C., Grun, S.,
Winklmair, A., Eisenreich, W., Bacher, A., Meeley, R.B., Briggs,
S.P., Simcox, K., and Gierl, A. (1997). Analysis of a chemical plant
defense mechanism in grasses. Science 277: 696–699.
Gamas, P., Niebel Fde, C., Lescure, N., and Cullimore, J. (1996). Use
of a subtractive hybridization approach to identify new Medicago
truncatula genes induced during root nodule development. Mol. Plant
Microbe Interact. 9: 233–242.
Goossens, A., Hakkinen, S.T., Laakso, I., Seppanen-Laakso, T.,
Biondi, S., De Sutter, V., Lammertyn, F., Nuutila, A.M., Soderlund,
H., Zabeau, M., Inze, D., and Oksman-Caldentey, K.M. (2003). A
functional genomics approach toward the understanding of second-
ary metabolism in plant cells. Proc. Natl. Acad. Sci. USA 100: 8595–
8600.
Grubb, C.D., Zipp, B.J., Ludwig-Muller, J., Masuno, M.N., Molinski,
T.F., and Abel, S. (2004). Arabidopsis glucosyltransferase UGT74B1
functions in glucosinolate biosynthesis and auxin homeostasis. Plant J.
40: 893–908.
Hansen, K.S., Kristensen, C., Tattersall, D.B., Jones, P.R., Olsen, C.
E., Bak, S., and Moller, B.L. (2003). The in vitro substrate regiospe-
cificity of recombinant UGT85B1, the cyanohydrin glucosyltransferase
from Sorghum bicolor. Phytochemistry 64: 143–151.
Haralampidis, K., Trojanowska, M., and Osbourn, A.E. (2002). Bio-
synthesis of triterpenoid saponins in plants. Adv. Biochem. Eng.
Biotechnol. 75: 31–49.
Haridas, V., Higuchi, M., Jayatilake, G.S., Bailey, D., Mujoo, K.,
Blake, M.E., Arntzen, C.J., and Gutterman, J.U. (2001). Avicins:
Triterpenoid saponins from Acacia victoriae (Bentham) induce apo-
ptosis by mitochondrial perturbation. Proc. Natl. Acad. Sci. USA 98:
5821–5826.
Hayashi, H., Huang, P., Kirakosyan, A., Inoue, K., Hiraoka, N.,
Ikeshiro, Y., Kushiro, T., Shibuya, M., and Ebizuka, Y. (2001).
Cloning and characterization of a cDNA encoding beta-amyrin syn-
thase involved in glycyrrhizin and soyasaponin biosyntheses in lico-
rice. Biol. Pharm. Bull. 24: 912–916.
Huhman, D.V., Berhow, M.A., and Sumner, L.W. (2005). Quantification
of saponins in aerial and subterranean tissues ofMedicago truncatula.
J. Agric. Food Chem. 53: 1914–1920.
Huhman, D.V., and Sumner, L.W. (2002). Metabolic profiling of sapo-
nins inMedicago sativa andMedicago truncatula using HPLC coupled
to an electrospray ion-trap mass spectrometer. Phytochemistry 59:
347–360.
Irmler, S., Schroder, G., St-Pierre, B., Crouch, N.P., Hotze, M.,
Schmidt, J., Strack, D., Matern, U., and Schroder, J. (2000). Indole
alkaloid biosynthesis in Catharanthus roseus: New enzyme activities
and identification of cytochrome P450 CYP72A1 as secologanin
synthase. Plant J. 24: 797–804.
Jorgensen, K., Rasmussen, A.V., Morant, M., Nielsen, A.H.,
Bjarnholt, N., Zagrobelny, M., Bak, S., and Moller, B.L. (2005).
Metabolon formation and metabolic channeling in the biosynthesis of
plant natural products. Curr. Opin. Plant Biol. 8: 280–291.
Kapusta, I., Janda, B., Stochmal, J., and Oleszek, W. (2005a).
Determination of saponins in aerial parts of barrel medic (Medicago
truncatula) by liquid chromatography-electrospray ionization/mass
spectrometry. J. Agric. Food Chem. 53: 7654–7660.
Kapusta, I., Stochmal, A., Perrone, A., Piacente, S., Pizza, C., and
Oleszek, W. (2005b). Triterpene saponins from barrel medic (Medi-
cago truncatula) aerial parts. J. Agric. Food Chem. 53: 2164–2170.
Kendall, W.A., and Leath, K.T. (1976). Effect of saponins on palatability
of alfalfa to meadow voles. Agron. J. 68: 473–476.
Kevei, Z., et al. (2007). 3-Hydroxy-3-methylglutaryl coenzyme a reduc-
tase 1 interacts with NORK and is crucial for nodulation in Medicago
truncatula. Plant Cell 19: 3974–3989.
Lange, B.M., and Ghassemian, M. (2003). Genome organization in
Arabidopsis thaliana: A survey for genes involved in isoprenoid and
chlorophyll metabolism. Plant Mol. Biol. 51: 925–948.
Leek, J.T., Monsen, E., Dabney, A.R., and Storey, J.D. (2006). EDGE:
Extraction and analysis of differential gene expression. Bioinformatics
22: 507–508.
Li, L., Cheng, H., Gai, J., and Yu, D. (2007). Genome-wide identification
and characterization of putative cytochrome P450 genes in the model
legume Medicago truncatula. Planta 226: 109–123.
Mackenzie, P.I., et al. (1997). The UDP glycosyltransferase gene
superfamily: Recommended nomenclature update based on evolu-
tionary divergence. Pharmacogenetics 7: 255–269.
Meesapyodsuk, D., Balsevich, J., Reed, D.W., and Covello, P.S.
(2007). Saponin biosynthesis in Saponaria vaccaria. cDNAs encoding
beta-amyrin synthase and a triterpene carboxylic acid glucosyltrans-
ferase. Plant Physiol. 143: 959–969.
Modolo, L.V., Blount, J.W., Achnine, L., Naoumkina, M.A., Wang, X.,
and Dixon, R.A. (2007). A functional genomics approach to (iso)
flavonoid glycosylation in the model legume Medicago truncatula.
Plant Mol. Biol. 64: 499–518.
Morita, M., Shibuya, M., Kushiro, T., Masuda, K., and Ebizuka, Y.
(2000). Molecular cloning and functional expression of triterpene
synthases from pea (Pisum sativum) new alpha-amyrin-producing
enzyme is a multifunctional triterpene synthase. Eur. J. Biochem. 267:
3453–3460.
Mylona, P., Owatworakit, A., Papadopoulou, K., Jenner, H., Qin, B.,
Findlay, K., Hill, L., Qi, X., Bakht, S., Melton, R., and Osbourn, A.
(2008). Sad3 and sad4 are required for saponin biosynthesis and root
development in oat. Plant Cell 20: 201–212.
Naoumkina, M., Farag, M.A., Sumner, L.W., Tang, Y., Liu, C.J., and
Dixon, R.A. (2007). Different mechanisms for phytoalexin induction by
pathogen and wound signals in Medicago truncatula. Proc. Natl.
Acad. Sci. USA 104: 17909–17915.
Oleszek, W. (1996). Alfalfa Saponins: Structure, Biological Activity, and
Chemotaxonomy. (New York: Plenum Press).
Oleszek, W., Junkuszew, M., and Stochmal, A. (1999). Determination
and toxicity of saponins from Amaranthus cruentus seeds. J. Agric.
Food Chem. 47: 3685–3687.
Osbourn, A.E. (2003). Saponins in cereals. Phytochemistry 62: 1–4.
Osbourn, A.E., and Field, B. (2009). Operons. Cell. Mol. Life Sci. 66:
3755–3775.
Peel, G.J., Pang, Y., Modolo, L.V., and Dixon, R.A. (2009). The LAP1
MYB transcription factor orchestrates anthocyanidin biosynthesis and
glycosylation in Medicago. Plant J. 59: 136–149.
Petit, P.R., Sauvaire, Y.D., Hillaire-Buys, D.M., Leconte, O.M.,
Genomics of Saponin Biosynthesis 865
Baissac, Y.G., Ponsin, G.R., and Ribes, G.R. (1995). Steroid sapo-
nins from fenugreek seeds: Extraction, purification, and pharmaco-
logical investigation on feeding behavior and plasma cholesterol.
Steroids 60: 674–680.
Price, K.R., Johnson, I.T., and Fenwick, G.R. (1987). The chemistry
and biological significance of saponins in foods and feedstuffs. Crit.
Rev. Food Sci. Nutr. 26: 27–135.
Qi, X., Bakht, S., Leggett, M., Maxwell, C., Melton, R., and Osbourn,
A. (2004). A gene cluster for secondary metabolism in oat: implica-
tions for the evolution of metabolic diversity in plants. Proc. Natl.
Acad. Sci. USA 101: 8233–8238.
Rajput, Z.I., Hu, S.H., Xiao, C.W., and Arijo, A.G. (2007). Adjuvant
effects of saponins on animal immune responses. J. Zhejiang Univ.
Sci. B 8: 153–161.
Ro, D.K., Arimura, G., Lau, S.Y., Piers, E., and Bohlmann, J. (2005).
Loblolly pine abietadienol/abietadienal oxidase PtAO (CYP720B1) is a
multifunctional, multisubstrate cytochrome P450 monooxygenase.
Proc. Natl. Acad. Sci. USA 102: 8060–8065.
Saito, K., Hirai, M.Y., and Yonekura-Sakakibara, K. (2008). Decoding
genes with coexpression networks and metabolomics - ’Majority
report by precogs’. Trends Plant Sci. 13: 36–43.
Shao, H., He, X., Achnine, L., Blount, J.W., Dixon, R.A., and Wang, X.
(2005). Crystal structures of a multifunctional triterpene/flavonoid
glycosyltransferase from Medicago truncatula. Plant Cell 17: 3141–
3154.
Shibuya, M., Hoshino, M., Katsube, Y., Hayashi, H., Kushiro, T., and
Ebizuka, Y. (2006). Identification of beta-amyrin and sophoradiol
24-hydroxylase by expressed sequence tag mining and functional
expression assay. FEBS J. 273: 948–959.
Shibuya, M., Katsube, Y., Otsuka, M., Zhang, H., Tansakul, P., Xiang,
T., and Ebizuka, Y. (2008). Identification of a product specific beta-
amyrin synthase from Arabidopsis thaliana. Plant Physiol. Biochem.
47: 26–30.
Shimura, K., et al. (2007). Identification of a biosynthetic gene cluster in
rice for momilactones. J. Biol. Chem. 282: 34013–34018.
Shulaev, V., Cortes, D., Miller, G., and Mittler, R. (2008). Metabol-
omics for plant stress response. Physiol. Plant. 132: 199–208.
Small, E. (1996). Adaptations to herbivory in alfalfa (Medicago sativa).
Can. J. Bot. 74: 807–822.
Sparg, S.G., Light, M.E., and van Staden, J. (2004). Biological activ-
ities and distribution of plant saponins. J. Ethnopharmacol. 94:
219–243.
Storey, J.D., and Tibshirani, R. (2003). Statistical significance for
genomewide studies. Proc. Natl. Acad. Sci. USA 100: 9440–9445.
Suzuki, H., Achnine, L., Xu, R., Matsuda, S.P., and Dixon, R.A. (2002).
A genomics approach to the early stages of triterpene saponin
biosynthesis in Medicago truncatula. Plant J. 32: 1033–1048.
Suzuki, H., Reddy, M.S.S., Naoumkina, M., Aziz, N., May, G.D.,
Huhman, D.V., Sumner, L.W., Blount, J.W., Mendes, P., and Dixon,
R.A. (2005). Methyl jasmonate and yeast elicitor induce differential
genetic and metabolic re-programming in cell suspension cultures of
the model legume Medicago truncatula. Planta 220: 698–707.
Suzuki, R., and Shimodaira, H. (2006). Pvclust: an R package for
assessing the uncertainty in hierarchical clustering. Bioinformatics 22:
1540–1542.
Tadege, M., Wen, J., He, J., Tu, H., Kwak, Y., Eschstruth, A., Cayrel,
A., Endre, G., Zhao, P.X., Chabaud, M., Ratet, P., and Mysore, K.S.
(2008). Large-scale insertional mutagenesis using the Tnt1 retrotrans-
poson in the model legume Medicago truncatula. Plant J. 54:
335–347.
Tava, A., and Odoardi, M. (1996). Saponins from Medicago Spp.:
Chemical characterization and biological activity against insects. Adv.
Exp. Med. Biol. 405: 97–109.
Thimm, O., Blasing, O., Gibon, Y., Nagel, A., Meyer, S., Kruger, P.,
Selbig, J., Muller, L.A., Rhee, S.Y., and Stitt, M. (2004). MAPMAN:
a user-driven tool to display genomics data sets onto diagrams
of metabolic pathways and other biological processes. Plant J. 37:
914–939.
Tusher, V.G., Tibshirani, R., and Chu, G. (2001). Significance analysis
of microarrays applied to the ionizing radiation response. Proc. Natl.
Acad. Sci. USA 98: 5116–5121.
Uematsu, Y., Hirata, K., and Saito, K. (2000). Spectrophotometric
determination of saponin in Yucca extract used as food additive. J.
AOAC Int. 83: 1451–1454.
Urbanczyk-Wochniak, E., and Sumner, L.W. (2007). MedicCyc: A
biochemical pathway database for Medicago truncatula. Bioinformat-
ics 23: 1418–1423.
Vincken, J.-P., Heng, L., de Groot, A., and Gruppen, H. (2007).
Saponins, classification and occurrence in the plant kingdom. Phyto-
chemistry 68: 275–297.
von Rad, U., Huttl, R., Lottspeich, F., Gierl, A., and Frey, M. (2001).
Two glucosyltransferases are involved in detoxification of benzoxazi-
noids in maize. Plant J. 28: 633–642.
Waller, G.R., and Yamasaki, K. (1996). Saponins Used in Traditional
and Modern Medicine. Advances in Experimental Medicine and
Biology. (New York: Plenum Press,).
Wang, N., Li, Z., Song, D., Li, W., Fu, H., Koike, K., Pei, Y., Jing, Y.,
and Hua, H. (2008). Lanostane-type triterpenoids from the roots of
Kadsura coccinea. J. Nat. Prod. 71: 990–994.
Werck-Reichhart, D., Bak, S., and Paquette, S. (2002). Cytochromes
P450. In The Arabidopsis Book, C.R. Somerville and E.M. Meyerowitz,
eds (Rockville, MD: American Society of Plant Biologists), doi/http://
www.aspb.org/publications/arabidopsis/.
Wilderman, P.R., Xu, M., Jin, Y., Coates, R.M., and Peters, R.J.
(2004). Identification of syn-pimara-7,15-diene synthase reveals func-
tional clustering of terpene synthases involved in rice phytoalexin/
allelochemical biosynthesis. Plant Physiol. 135: 2098–2105.
Woo, H.H., Orbach, M.J., Hirsch, A.M., and Hawes, M.C. (1999).
Meristem-localized inducible expression of a UDP-glycosyltransfer-
ase gene is essential for growth and development in pea and alfalfa.
Plant Cell 11: 2303–2315.
Yonekura-Sakakibara, K., Tohge, T., Matsuda, F., Nakabayashi, R.,
Takayama, H., Niida, R., Watanabe-Takahashi, A., Inoue, E., and
Saito, K. (2008). Comprehensive flavonol profiling and transcriptome
coexpression analysis leading to decoding gene-metabolite correla-
tions in Arabidopsis. Plant Cell 20: 2160–2176.
Yonekura-Sakakibara, K., Tohge, T., Niida, R., and Saito, K. (2007).
Identification of a flavonol 7-O-rhamnosyltransferase gene determin-
ing flavonoid pattern in Arabidopsis by transcriptome coexpression
analysis and reverse genetics. J. Biol. Chem. 282: 14932–14941.
Zar, J.H. (1999). Biostatistical Analysis. (Upper Saddle River, NJ:
Prentice Hall).
866 The Plant Cell
DOI 10.1105/tpc.109.073270; originally published online March 26, 2010; 2010;22;850-866Plant Cell
Lloyd W. Sumner and Richard A. DixonMarina A. Naoumkina, Luzia V. Modolo, David V. Huhman, Ewa Urbanczyk-Wochniak, Yuhong Tang,
Medicago truncatulaBiosynthesis in Genomic and Coexpression Analyses Predict Multiple Genes Involved in Triterpene Saponin
This information is current as of December 5, 2018
Supplemental Data /content/suppl/2010/03/15/tpc.109.073270.DC1.html
References /content/22/3/850.full.html#ref-list-1
This article cites 74 articles, 21 of which can be accessed free at:
Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X
eTOCs http://www.plantcell.org/cgi/alerts/ctmain
Sign up for eTOCs at:
CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain
Sign up for CiteTrack Alerts at:
Subscription Information http://www.aspb.org/publications/subscriptions.cfm
is available at:Plant Physiology and The Plant CellSubscription Information for
ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists