Genomic and Coexpression Analyses Predict Multiple Genes ...Genomic and Coexpression Analyses...

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 Botânica, Instituto de CienciasBiologicas, Universidade Federal de Minas Gerais, Av. Antônio 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


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 probesets 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 SupplementalData 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.


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 identicalshapes 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

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/Kmratio 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


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

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

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


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. Tandemmass 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


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.

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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

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Genomic and Coexpression Analyses Predict Multiple Genes ...Genomic and Coexpression Analyses...

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