A genomics approach to the early stages of triterpenesaponin biosynthesis in Medicago truncatula

Hideyuki Suzuki1,�, Lahoucine Achnine1,�, Ran Xu2, Seiichi P. T. Matsuda2 and Richard A. Dixon1,�

1Plant Biology Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, and2Departments of Chemistry and Biochemistry and Cell Biology, Rice University, Houston, TX 77005, USA

Received 11 July 2002; revised 20 September 2002; accepted 27 September 2002.�For correspondence (fax þ1 580 224 6692; e-mail radixon@noble.org).�These authors contributed equally to this work.

Summary

The saponins of the model legume Medicago truncatula are glycosides of at least five different triterpene

aglycones: soyasapogenol B, soyasapogenol E, medicagenic acid, hederagenin and bayogenin. These agly-

cones are most likely derived from b-amyrin, a product of the cyclization of 2,3-oxidosqualene. Mining

M. truncatula EST data sets led to the identification of sequences putatively encoding three early enzymes

of triterpene aglycone formation: squalene synthase (SS), squalene epoxidase (SE), and b-amyrin synthase

(b-AS). SS was functionally characterized by expression in Escherichia coli, two forms of SE by comple-

mentation of the yeast erg1 mutant, and b-AS by expression in yeast. b-Amyrin was the sole product of the

cyclization of squalene epoxide by the recombinant M. truncatula b-AS, as judged by GC–MS and NMR.

Transcripts encoding b-AS, SS and one form of SE were strongly and co-ordinately induced, associated with

accumulation of triterpenes, upon exposure of M. truncatula cell suspension cultures to methyl jasmonate.

Sterol composition remained unaffected by jasmonate treatment. Molecular verification of induction of the

triterpene pathway in a cell culture system provides a new tool for saponin pathway gene discovery by DNA

array-based approaches.

Keywords: triterpene, sterol, saponin, methyl jasmonate, elicitation, functional expression.

Introduction

Triterpene glycoside saponins are attracting increasing

interest in view of their multiple biological activities. These

both positively and negatively impact plant traits. Thus,

whereas some saponins display allelopathic (Waller et al.,

1993), antimicrobial (Nagata et al., 1985; Osbourn, 1996;

Papadopoulou et al., 1999), and anti-insect (Pedersen et al.,

1976; Tava and Odoardi, 1997) activity, they can also be

toxic to monogastric animals, act as antipalatability factors,

or negatively impact forage digestibility in ruminants

(Cheeke, 1976; Oleszek, 1997; Oleszek et al., 1999; Small,

1996). Other saponins have potentially useful pharmacol-

ogical activities, including anticholesterolemic (Cheeke,

1976), anticancer (Haridas et al., 2001; Park et al., 2001),

adjuvant (Behboudi et al., 1999; Marciani et al., 2000), and

hemolytic (Jones and Elliott, 1969) activity. Despite the

obvious interest in facilitating or inhibiting production of

triterpene saponins for crop improvement or develop-

ment of pharmacological agents, most of the steps in their

biosynthesis remain uncharacterized at the molecular

level.

We have chosen the model legume Medicago truncatula

as a suitable species for a functional genomic approach to

triterpene saponin biosynthesis in view of the availability of

extensive EST resources (Bell et al., 2001) and the interest-

ing saponin profile of this species (Huhman and Sumner,

2002). Metabolic profiling of M. truncatula roots using

reverse-phase HPLC and electrospray ionization mass spec-

trometry showed the presence of a more complex mixture

of triterpenes than found in the closely related and pre-

viously well-studied species alfalfa (M. sativa) (Massiot

et al., 1988; Oleszek and Jurzysta, 1990; Oleszek et al.,

1992; Tava et al., 1993). Five different triterpene aglycones,

soyasapogenol B, soyasapogenol E, medicagenic acid,

hederagenin, and bayogenin were found to be the core

of the 37 M. truncatula saponins identified (Huhman and

Sumner, 2002). All these aglycones are most likely derived

from b-amyrin, the initial product of cyclization of 2,3-oxi-

dosqualene, as shown in Figure 1.

To utilize the genomic resources of M. truncatula for

gene discovery in the saponin pathway, it is necessary to

The Plant Journal (2002) 32, 1033–1048

� 2002 Blackwell Publishing Ltd 1033

Figure 1. The biosynthesis of b-amyrin and cycloartenol, and the involvement of cytochrome P450 and glycosyltransferase enzymes in the biosynthesis of the triterpene aglycones and selected conjugates found inMedicago truncatula.

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functionally characterize one or more saponin biosynthetic

genes prior to DNA array-based approaches. The first com-

mitted step in triterpene biosynthesis in Medicago is cata-

lyzed by a specific oxidosqualene cyclase (OSC), b-amyrin

synthase (b-AS). In higher plants, oxidosqualene is a pre-

cursor common to the biosynthesis of both steroids and

triterpenoids (Abe et al., 1993). In sterol biosynthesis in

animals and fungi, the cyclization of 2,3-oxidosqualene

leads to the formation of lanosterol, whereas cycloartenol

is the first cyclized sterol precursor in plants. b-AS has been

functionally characterized from Panax ginseng (Kushiro

et al., 1998), pea (Morita et al., 2000) and Arabidopsis

thaliana (Husselstein-Muller et al., 2001), and is closely

related to plant cycloartenol synthase, which has also been

cloned and functionally characterized (Corey et al., 1993;

Hayashi et al., 2000). Surprisingly, a recently characterized

monocot b-AS from oat is phylogenetically distinct from

dicot b-AS enzymes (Haralampidis et al., 2001). b-AS may

produce one or more products from the cyclization of 2,3-

oxidosqualene, depending on the plant source (Abe et al.,

1993; Husselstein-Muller et al., 2001; Kushiro et al., 1998,

2000).

The two enzymes preceding OSC, namely squalene

synthase (SS) and squalene epoxidase (SE), have been well

characterized in mammals and yeast (Jandrositz et al.,

1991; Laden et al., 2000; Lee et al., 2000; Pandit et al.,

2000). SS has been functionally characterized from Arabi-

dopsis (Kribii et al., 1997; Nakashima et al., 1995). However,

although plant SE genes have been annotated based upon

sequence similarity to the mammalian and yeast enzymes

(Schafer et al., 1999), plant SE has not been functionally

characterized. SE is membrane associated, requires NADPH

cytochrome P450 reductase and, in mammals, additional

soluble protein factors for its activity (Laden et al., 2000;

Shibata et al., 2001). It is not known whether specific forms

of SS and SE might be differentially associated with sterol

and triterpene biosynthesis in plants.

We here describe an EST data mining approach for iso-

lation of candidate SS, SE and b-AS genes from M. trunca-

tula, and the functional identification of these genes by

heterologous expression in E. coli or yeast. The corre-

sponding cDNA sequences have been used as probes for

development of an inducible cell culture system for triter-

pene pathway gene discovery.

Results

Identification of early triterpene pathway genes in

M. truncatula by mining EST data sets

Candidate ESTs for the first steps of the saponin biosyn-

thetic pathway in M. truncatula were identified by mining

publicly available EST data sets representing cDNA

libraries from a variety of different organs and biotic/

abiotic treatments (Table 1). To obtain full-length mRNA

sequences for the putative SS, SE and b-AS genes, we

retrieved and analyzed EST clones found in cDNA libraries

from M. truncatula roots, nodulated roots, stems, cell sus-

pension cultures and leaves, and from some of the above

tissues following treatments such as insect damage, elici-

tation with yeast extract, drought, or phosphate starvation.

In the TIGR M. truncatula Gene Index (MtGI) database

(Quackenbush et al., 2000; http://www.tigr.org/tdb/mtgi/),

the putative SS ESTs were clustered into one tentative

consensus, whereas putative SE and b-AS ESTs were each

clustered into three tentative consensuses (Table 1). Two

SE genes, SE1 and SE2, are each present in a single copy

in the M. truncatula genome, as shown by DNA gel blot

analysis in Figure 2(b,c). Neither SE1 nor SE2 has restriction

Table 1 Medicago truncatula EST clones and tentative consensus (TC) sequences annotated as squalene synthase (SS), squaleneepoxidase (SE), and b-amyrin synthase (b-AS)

Gene nameTentative consensus (TC)sequence numbers

Number of EST clones per TC (number of full-length clones)[library distribution (number of clones per library)]

Squalene synthase (SS) �TC35874 12 (7) [a(3), b(2), e(1), f(2), �g(4)]�TC28416 7 (4) [�c(1), d(1), f(1), g(2), h(1), l(1)]

Squalene epoxidase (SE) TC29021 3 (0) [c(1), g(2)]�TC37711 2 (1) [�f(2)]TC28833 3 (0) [e(1), j(2)]

b-amyrin synthase (b-AS) TC28834 2 (0) [b(1), c(1)]�TC28878 8 (3) [b(1), c(1), e(1),�g(5)]

Data are from cDNA libraries from a number of different tissue sources sequenced at the Samuel Roberts Noble Foundation. Further detailscan be found in the TIGR M. truncatula Gene Index (MtGI) at (http://www.tigr.org/tdb/mtgi/). TC sequences are assembled from ESTs, andmay represent full-length transcripts. TC annotations contain information on the source library and abundance of ESTs. The tissue sourcesof the cDNA libraries are: a, root (6593); b, stem (10 314); c, developing leaf (7831); d, phosphate-starved leaf (9034); e, drought-inducedwhole plants (8416); f, yeast-elicited cell culture (8926); g, insect-damaged leaf (9921); h, developing flower (3404); i, nodulated root (29 721);and j, germinating seed (451). The numbers in brackets refer to the total number of ESTs sequenced in each library as of December 2001.The symbol (�) denotes the library from which the full-length clones used in the present work were isolated, and the corresponding TC.

� Blackwell Publishing Ltd, The Plant Journal, (2002), 32, 1033–1048

Triterpene saponin biosynthesis 1035

sites for BamHI, SalI, or XbaI. EcoRI cuts once in SE2, but

does not cut SE1; thus, the common major band in the

EcoRI lanes in Figure 2(b,c) is likely due to cross-hybridiza-

tion with the other SE gene. SE1 and SE2 share 76.6%

sequence identity at the nucleic acid level. b-AS and SS

are both present in two copies in M. truncatula (Figure 2a,d).

DNA sequence alignment using the Clustal program

indicated that M. truncatula (GenBank Accession #

AJ430610) and soybean (Glycine max) SS proteins are

closely related (88.6% at the amino acid level), as would

be expected, since both species belong to the Fabaceae.

M. truncatula SE1 (GenBank Accession # AJ430609) and

SE2 (GenBank Accession # AJ430608) proteins are more

closely related to P. ginseng putative SE, with 77.1 and

74.4% sequence identity, respectively, than to Arabidopsis

and Brassica SEs (Figure 3a). The two distinct types of plant

OSC, cycloartenol synthase and b-AS, exhibit a relatively

high level of amino acid sequence identity, even though

their reaction products are quite distinct (Hayashi et al.,

2000; Kushiro et al., 1998). Alignment of known plant

cycloartenol synthase and b-AS proteins indicates that

the putative M. truncatula b-AS (GenBank Accession #

AJ430607) falls into the b-amyrin synthase group (Fig-

ure 3c). M. truncatula b-AS protein was closely related to

pea (Pisum sativum) b-AS with 94.7% sequence identity.

Figure 3(b) shows amino acid sequence alignments of

M. truncatula SE1 and SE2 with the enzymes from human,

rat, and yeast. There is a high degree of sequence con-

servation in certain key regions, particularly around the

squalene and FAD binding domains. The M. truncatula

Figure 2. DNA gel blot analysis of triterpene pathway genes in Medicago truncatula.Genomic DNA was cut with the enzymes shown (B¼BamHI, S¼SalI, E¼EcoRI, X¼XbaI), fragments resolved by agarose gel electrophoresis, and blots probedwith cDNAs encoding squalene synthase (a), squalene epoxidase 1 (b), squalene epoxidase 2 (c), and b-amyrin synthase (d). Molecular size markers (in Kb) areshown on the left.

Figure 3. Sequence analysis of Medicago truncatula SE and b-AS genes.(a) Dendrogram comparison of the sequences of oxidosqualene cyclases. The dendrogram was created using the Clustal W and TreeViewPPC programs. Key andGenBank accession numbers of the oxidosqualene cyclase superfamily: bAS, b-amyrin synthase: M. truncatula bAS (this study), A. strigosa bAS (AsbAS,AJ311789), P. ginseng bAS (PgAbAS, B009030); G. glabra bAS (GgbAS, AB037203); P. sativum bAS (PsbAS, AB034802). LS, lanosterol synthases: Homo sapiensLS (U22526); Rattus norvegicus LS (U31352); Saccharomyces cerevisiae LS (U04841). SHC, squalene-hopene cyclase: Alicyclobacillus acidocaldarius SHC(AB007002). LuS, lupeol synthases: Arabidopsis thaliana LuS (AtLuS, AAD05032); O. europaea LuS (OeLuS, AB025343); Taraxacum officinale LuS (ToLuS,AB025345). CAS, cycloartenol synthases: M. truncatula CAS (this study), A. strigosa CAS (AsCAS, AJ311790); O. sativa CAS (OsCAS, AF169966); Luffa cylindricaCAS (LcCAS, AB033334); G. glabra CAS (GgCAS, AB025968); P. sativum CAS (PsCAS, D89619); P. ginseng CAS (PgCAS, AB009029); O. europaea CAS (OeCAS,AB025344).(b) Amino acid sequence alignments of M. truncatula putative squalene epoxidases 1 and 2 with published squalene epoxidases from other organisms. Thehighly conserved squalene and FAD binding domains are highlighted with boxes in the N- and C-terminal portions of the protein, respectively. The arrow marksthe position in the N-terminus at which the M. truncatula proteins were truncated for expression in yeast.(c) Dendrogram comparing the sequences of squalene epoxidases. The dendrogram was created using the Clustal W and TreeViewPPC programs. Key andGenBank accession numbers: Arabidopsis thaliana SE1 (AtSE1), A. thaliana SE2 (AtSE2), A. thaliana SE3 (AtSE3), A. thaliana SE4 (AtSE4, AJ005930); A. thalianaSE5 (AtSE5, AJ005927); A. thaliana SE6 (AtSE6, AJ005929); Brassica napus SE1 (BnSE1, AJ005931), B. napus SE2 (BnSE2, AJ005928), M. truncatula SE1 (thisstudy), M. truncatula SE2 (this study), rat SE (D37920), human SE (D78130), yeast erg1 (M64994), Candida SE (D88252).

� Blackwell Publishing Ltd, The Plant Journal, (2002), 32, 1033–1048

1036 Hideyuki Suzuki et al.

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Triterpene saponin biosynthesis 1037

enzymes are more similar to the mammalian enzymes than

to the enzyme from yeast.

Transcript abundance of early saponin pathway genes in

M. truncatula

Abundance of putative saponin biosynthetic enzyme tran-

scripts in different organs was first assessed in silico from

analysis of EST occurrence in the various cDNA libraries

using the data available in the TIGR M. truncatula Gene

Index database (Table 1). Putative triterpene pathway genes

appeared to be expressed at a higher level in insect-

damaged leaves than in control leaves based on relative

EST abundance (Table 1). Three SS full-length clones

(including the one functionally characterized below) and

one truncated clone were found among the 9921 clones

sequenced from the insect-damaged leaf library, but only

one truncated SE clone (NF026F08IN) was found. Two full-

length and two truncated b-AS clones were found, includ-

ing the one functionally characterized below. The highest

abundance was five ESTs for b-AS from the insect-

damaged leaf library.

The essential features of the in silico transcript abun-

dance analysis were confirmed by RNA gel blot analysis

in several cases, using RNA samples from the original

preparations used for cDNA library construction (Figure 4).

To distinguish between SE1 and SE2 transcripts, N-terminal

gene-specific probes were generated corresponding to the

50-untranslated regions and the open reading frames 50 of

the FAD binding domains. Putative SS transcripts were

remarkably abundant in roots, whereas flower, leaf, petiole,

cell culture, and stem showed a lower level of expression.

The analysis of M. truncatula SE1 transcripts shows that

this gene is expressed weakly in all tissues examined.

Exposure of M. truncatula cell suspension cultures to yeast

elicitor for 24 h resulted in an enhancement of SS, but not

SE1, transcripts. SE2 transcripts were also expressed in all

tissues examined, but at higher levels than SE1 transcripts

and with evidence of weak induction in cell cultures by

yeast elicitor. Putative b-AS transcripts were most highly

expressed in flower and root and induced from a very low

basal level in yeast-elicited cell cultures.

Functional characterization of M. truncatula squalene

synthase

SS catalyzes the reductive dimerization of two molecules

of farnesyl diphosphate (FPP) in a two-step reaction to

Figure 4. RNA gel blot analysis of tissue distribution of Medicago truncatula triterpene pathway transcripts.(a) Total RNA was isolated from the tissues shown, resolved by agarose gel electrophoresis, blotted, and probed with full-length M. truncatula squalene synthase(SS), N-terminal fragments from squalene epoxidase 1 (SE1) and squalene epoxidase 2 (SE2), and full-length b-amyrin synthase (b-AS) cDNAs. Cell suspensioncultures were of root origin and were induced with yeast elicitor (YE).(b) Quantitation of the data in (a). Relative transcript abundance was calculated by dividing the intensity of each transcript to that of the corresponding 18S RNAtranscript level. C¼ cell culture, C(E)¼ elicited cell culture, F¼ flower, L¼ leaf, P¼peptide, R¼ root, S¼ stem.

� Blackwell Publishing Ltd, The Plant Journal, (2002), 32, 1033–1048

1038 Hideyuki Suzuki et al.

produce squalene. This reaction is believed to proceed via

head-to-head coupling of two FPP molecules to form squa-

lene via a stable cyclopropylcarbinyl diphosphate inter-

mediate (Pandit et al., 2000). Functional expression of the

M. truncatula putative SS was accomplished in E. coli BL21

cells. SDS-PAGE analysis of total proteins showed that a 43-

kDa band, corresponding to the predicted size of the recom-

binant protein, appeared in extracts from IPTG induced

E. coli, but not in cultures harboring an empty pET-15b

vector (data not shown). To demonstrate squalene

synthase activity, harvested cells were disrupted, centri-

fuged, and the supernatant used for enzyme assay in the

presence of 14C-FPP, NADPH and Mg2þ. Reverse-phase

TLC was used to separate the product(s). No 14C-squalene

product is formed in extracts of E. coli transformed with

the empty vector (Figure 5a). In contrast, extracts from

E. coli transformed with pET15b containing recombinant

SS (pET-SS), in spite of the very small proportion of

soluble recombinant enzyme, catalyzed formation of a

labeled product that co-migrated with authentic squalene.

When NADPH was omitted, no squalene product was

observed. A strong reduction in squalene formation was

also observed on omitting Mg2þ from the incubation mix-

ture, the residual activity perhaps being supported by

endogenous Mg2þ.

We further characterized the M. truncatula SS reaction by

substituting Mg2þ in the incubation mixture with other

divalent cations. Mn2þ, Co2þ, and Fe2þ could substitute

for Mg2þ as cofactors in this reaction, whereas Ca2þ,

Cu2þ, or Zn2þcould not (Figure 5a).

Functional characterization of M. truncatula squalene

epoxidase

SE catalyzes the insertion of an oxygen atom across a

carbon–carbon double bond to form an epoxide in a

reaction more typical of P450-type reactions. Squalene

monoxygenases have been cloned and functionally

characterized from yeast, rat, and human (Jandrositz

et al., 1991; Laden et al., 2000; Sakakibara et al., 1995),

but not from plants. SE, encoded by the ERG1 gene in

yeast, is a key enzyme in the sterol biosynthetic pathway.

The KLN1 strain of yeast (MATa, erg1::URA3, leu2, ura3,

trp1) used here for the functional characterization of puta-

tive M. truncatula SE is an obligate ergosterol auxotroph;

disruption of ERG1 is lethal unless ergosterol is supplied to

cells growing under anaerobic conditions (Landl et al.,

1996).

To functionally characterize the two putative M. trunca-

tula squalene epoxidases, the SE1 and SE2 coding

sequences, with 47 amino acids truncated from the N-

teminus of SE1, and 52 amino acids truncated from the

N-teminus of SE2, and the ERG1 ORF as a positive control

were cloned into the pWV3 vector, containing the LEU2

selectable marker, under the control of the constitutive

pADH1 promoter. The N-terminal truncation sites were

chosen by comparison with the yeast protein, which has

a short N-terminus compared to plant or mammalian SE

(Figure 3b); we thought that this truncation might therefore

facilitate expression of the plant enzyme in yeast. Selection

of transformants for the Leuþ phenotype was made in SD

medium supplied with ergosterol and tryptophan under

anaerobic conditions, as shown in Figure 5(b), panel 1.

As expected, KLN1 did not grow because the medium

was deprived of Leu (Landl et al., 1996). When plated in

YPD (or SDþ trp) medium without ergosterol under anae-

robic conditions, the transformants were not viable (Fig-

ure 5b, panel 2), whereas under aerobic conditions, they

exhibited strong growth (Figure 5b, panel 3). pWV3 trans-

formants were not able to grow under either condition,

showing that the SE or ERG1 (positive control) inserts

contributed to this growth. Thus, the growth of the trans-

formants is oxygen dependent, as is the SE reaction. These

data show that the ergosterol biosynthetic pathway in the

yeast erg1 knockout could be re-constituted by heterolo-

gous complementation with M. truncatula SE with or with-

out truncation of the N-terminus.

Functional characterization of M. truncatula b-amyrin

synthase

The enzyme encoded by EST NF051E06IN, when expressed

in yeast, cyclized oxidosqualene to form product(s) that co-

migrated with b-amyrin on TLC (data not shown), whereas

the yeast strain SMY8 harboring the empty vector did not

form this compound(s). GC analysis revealed that the

recombinant M. truncatula b-AS only produced a single

cyclization product from oxidosqualene, as shown in Fig-

ure 5(c). The GC relative retention time (Rt) of the cyclization

product, TMS ether, was identical to that of authentic b-

amyrin TMS ether (Rt¼ 1.23, relative to epicoprostanol

TMS ether). The mass spectra (MS) of the enzymatic pro-

duct, b-amyrin standard and their co-injection agreed with

each other. (EI-MS): (TMS ether) m/z¼ 498 [M]þ (6%), 483

[M-CH3]þ (3%), 408 [M-Me3SiOH]þ (2%), 393 [M-Me3SiOH-

CH3]þ (3%), 218 (C-ring fragment, 100%), 203 [m/z 218 –

CH3]þ (39%). NMR data further confirmed the identification

of b-amyrin. Key 1H-NMR signals of the authentic sample

matched those of the NF051E06IN product to �0.001 p.p.m.

(500 MHz, CDCl3, tetramethylsilane as internal standard): d5.184 (t, 3.5 Hz, 1H, H-12), 3.223 (ddd, 11.2, 6.0, 4.7 Hz, 1H, H-

3), 1.135 (d, 1.0Hz, 3H, H-27), 0.998 (s, 3H, H-23), 0.968 (s, 3H,

H-26), 0.938 (s, 3H, H-25), 0.872 (s, 6H, H-29, H-30), 0.832 (s,

3H, H-28), 0.792 (s, 3H, H-24), 0.742 (d, 11.7, 1.9 Hz, 1H, H-5).

The observed MS and NMR data agreed with literature

values for b-amyrin (Segura et al., 2000). A 290-ml yeast

culture produced 1.7 mg of b-amyrin (>98% pure) from

14 mg of oxidosqualene substrate.

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Triterpene saponin biosynthesis 1039

� Blackwell Publishing Ltd, The Plant Journal, (2002), 32, 1033–1048

1040 Hideyuki Suzuki et al.

Development of an inducible cell culture system for

functional genomics approaches to further understand

triterpene saponin biosynthesis in M. truncatula

The reactions of triterpene biosynthesis beyond the initial

cyclization step catalyzed by b-AS are complex, and none of

the enzymes involved in Medicago has been characterized

at the molecular level. However, it is possible to present a

putative pathway based on the nature of the saponins

identified in M. truncatula and analogies with parallel oxi-

dative reactions involved in brassinosteroid biosynthesis

(Bishop et al., 1999; Fujioka et al., 2000; Huhman and

Sumner, 2002; Noguchi et al., 2000). It is likely that the

oxidative reactions are catalyzed by a series of cytochrome

P450 monooxygenases, with the final reactions catalyzed

by glycosyltransferases (GTs), as outlined in Figure 1. To

utilize existing information on P450s and GTs from the

M. truncatula genomic databases, it is necessary to develop

a system in which the saponin pathway can be rapidly and

reproducibly induced as a basis for micro- and macro-array

experiments.

Extractation and quantitation of the multiple M. trunca-

tula triterpene saponins is not trivial (Huhman and Sumner,

2002), and is therefore not the best assay method for

determining expression of the triterpene pathway. We

therefore decided to measure changes in transcript levels

by RNA gel blot analysis, using the functionally confirmed

Medicago b-AS, SE, and SS as probes, in a series of experi-

ments designed to investigate conditions for inducing tri-

terpene synthesis in M. truncatula root cell suspension

cultures. We tested a variety of potential elicitors, including

methyl jasmonate (MeJA), yeast elicitor (YE), salicylic acid

(SA), and abscisic acid (ABA), for their ability to induce

saponin pathway transcripts in the cultures. YE weakly

induced SS, SE2, and b-AS, as previously shown in Figure 4.

Enhancement of b-AS transcript levels was two- and sixfold

at 12 h post-elicitation with YE and SA, respectively (data

not shown). b-AS transcripts were induced to a maximum

level of 2.5-fold 1 h after exposure to ABA (data not shown).

Strongest elicitation of b-AS transcripts was found with

MeJA, which induced an increase of up to 30-fold by 8–

24 h post-elicitation, as shown in Figure 6(a,b). SS tran-

scripts were coordinately induced with b-AS transcripts

in response to MeJA. SE1 transcripts were not significantly

induced by MeJA, whereas SE2 transcript induction closely

followed that of b-AS (Figure 6a,b), indicating that SE2, but

not SE1, may function specifically in the formation of

triterpenoids. In contrast, elicitation with MeJA caused a

significant reduction in cycloartenol synthase transcript

levels.

Treatment of cell cultures with MeJA was confirmed to

induce accumulation of triterpene saponins, as assessed by

chemical extraction and analysis by LC/MS (Figure 7a).

Small amounts of hederagenin glycoside were present in

unelicited cultures. These increased approximately 10-fold

by 24 h post-elicitation, and glycosides of soyasapogenols

B and E appeared. The latter compounds were not detected

in the unelicited cultures.

The reduction in cycloartenol synthase transcripts in

response to MeJA could indicate preferential channeling

of oxidosqualene from biosynthesis of housekeeping ster-

ols into the triterpene pathway. However, GC/MS analysis

of control and elicited cell cultures revealed no significant

change in sterol profiles at 24 h post-elicitation, as shown in

Figure 7(b). The major difference between elicited and con-

trol cultures was the appearance, following elicitation, of a

small peak shown to correspond to the trimethylsilyl ether

of b-amyrin.

MeJA has been suggested to be a signal molecule for the

biosynthesis of phytoalexins (Gundlach et al., 1992). In

Medicago species, the phytoalexins are isoflavonoids

derived from the phenylpropanoid/flavonoid pathway

(Dixon, 1999). To determine whether the phenylpropanoid

pathway is co-induced with the triterpene biosynthetic

pathway following exposure of cells to MeJA, membranes

were probed with labeled M. truncatula phenylalanine

ammonia lyase (PAL) and chalcone synthase (CHS) cDNAs.

PAL transcripts were only weakly induced by MeJA, with a

Figure 5. Functional characterization of Medicago truncatula squalene synthase, squalene epoxidase, and b-amyrin synthase.(a) M. truncatula squalene synthase (SS) was expressed in Escherichia coli BL21(DE3, pLyS) using the pET-15b expression vector. The enzyme was assayed byradio-TLC as described in Experimental procedures. Lane 1: extract from E. coli harboring pET-15b empty vector assayed in the presence ofNADPHþMgCl2þDTTþKFþ 14C-FPPþ50 mM Tris–HCl (pH 7.6) (negative control). Lane 2: extract from E. coli harboring pET-SS assayed as in lane 1 (positivecontrol). Lanes 3–11: extracts from E. coli harboring pET-SS assayed with different components in the reaction mixture. Lane 3: without NADPH; lane 4, withoutDTT; lane 5, without MgCl2; lanes 6–11, MnCl2, CaCl2, CoCl2, CuCl2, FeCl2, ZnCl2 in place of MgCl2; lane 12, authentic 14C-squalene. SQ, squalene; FOH, farnesol.(b) Complementation of the yeast erg1 mutant by M. truncatula squalene epoxidase. (1) Selection of transformants for the Leuþ phenotype in SD mediumsupplied with ergosterol and tryptophan under anaerobic conditions. (2) Plating of yeast cells in YPD (or SDþ trp) medium without ergosterol under anaerobicconditions. The transformants were not viable. The same result was obtained with SD medium plus tryptophan. (3) Growth of yeast cells in YPD medium withoutergosterol under aerobic conditions. KLN1¼non-transformed KLN1 yeast strain; pWV3¼KLN1 yeast transformed with the pWV3 yeast expression vector only;pWV3-SE1 and pWV3-SE2¼KLN1 yeast transformed with the pWV3 yeast expression vector containing SE1 and SE2 ORFs, respectively; pWV3-SE1D47 andpWV3-SE2D52¼KLN1 yeast transformed with the pWV3 yeast expression vector containing SE1 and SE2, with 47 and 52 amino acids truncated from the N-termini, respectively.(c) Functional expression of M. truncatula b-amyrin synthase in yeast. Gas chromatogram showing the product from the incubation of recombinant b-amyrinsynthase with oxidosqualene. A single product, representing the TMS ether of b-amyrin, was produced. The internal standard peak is the TMS ether ofepicoprostanol.

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Triterpene saponin biosynthesis 1041

Figure 6. Differential induction of triterpene, sterol, and phenylpropanoid pathway transcripts in Medicago truncatula cell suspension cultures exposed toMeJA.(a) Total RNA was isolated from elicited cell cultures at the various times shown, resolved by agarose gel electrophoresis, blotted and hybridized withM. truncatula squalene synthase (SS), squalene epoxidase 1 (SE1), squalene epoxidase 2 (SE2), b-amyrin synthase (b-AS), cycloartenol synthase (CAS),phenylalanine ammonia-lyase (PAL), and chalcone synthase (CHS) cDNAs. The SE probes were N-terminal fragments that distinguished between SE1 and SE2.18S rRNA was probed as a control for equal loading and transfer of RNA.(b) Blots were quantified by phosphorimager analysis, and data plotted with normalization to the zero time value as 100%.

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1042 Hideyuki Suzuki et al.

maximum increase of only 1.5-fold at 24 h post-elicitation.

More strikingly, CHS transcript levels decreased in parallel

to the increase in b-AS mRNA (Figure 6a,b).

Discussion

Mining M. truncatula EST data sets yielded candidate

clones for the enzymes that catalyze the early steps of

triterpene biosynthesis, namely SS, SE, and b-AS. Several

of the ESTs proved to originate from full-length clones,

which were functionally characterized by heterologous

expression. Divalent cation-dependent production of squa-

lene from farnesyl diphosphate in E. coli extracts provided

functional confirmation for heterologous expression of

M. truncatula SS. A. thaliana SS has been functionally

expressed, and shown to produce squalene in the presence

of Mg2þ and NADPH, and dehydrosqualene in the presence

of Mn2þ but absence of NADPH (Nakashima et al., 1995).

The M. truncatula squalene synthase could use Mn2þ or

Mg2þ equally well as cofactors for squalene formation in

the presence of NADPH. Interestingly, the intact full-length

Arabidopsis SS1 cannot complement a yeast SS mutant,

even though the yeast cells expressing the Arabidopsis

enzyme contain detectable SS activity when assayed in vi-

tro. This has been shown to be due to a requirement for a

specific C-terminal portion of the yeast SS for metabolic

channeling of squalene through the yeast sterol pathway

(Kribii et al., 1997). This is an interesting feature from the

point of view of the organization of potential metabolic

complexes necessary for channeling of squalene into either

the triterpene or the sterol pathway in plants. The fact that

M. truncatula possesses two isoforms of the subsequent

enzyme in the pathway, SE1 and SE2, raises the question of

whether these may have different biochemical functions in

relation to triterpene and sterol biosynthesis. This idea is

supported by the co-induction of SE2, but not SE1, with b-

AS in MeJA-treated cell cultures.

Mammalian SE plays a pivotal role in cholesterol bio-

synthesis, and the enzyme is expressed at low levels in

most tissues (Ono and Bloch, 1975; Yamamoto and Bloch,

1970). Detailed enzymological characterization of human

SE has been reported (Laden et al., 2000). In yeast, the

squalene epoxidase Erg1p exhibits dual localization in the

endoplasmic reticulum and in lipid particles (Leber et al.,

1998). Although plant genes with sequence similarity to

mammalian SE have been described in the literature, the

present report is, to the best of our knowledge, the first to

document the functional characterization of a plant SE. The

two isoforms of M. truncatula squalene epoxidase, SE1 and

SE2, share 82.1% amino acid identity. Both M. truncatula

SEs could complement the ergosterol biosynthetic path-

way in the Erg1 knockout yeast strain KLN1. This is inter-

esting in view of the failure of Arabidopsis SS to correctly

couple with the sterol biosynthetic machinery in yeast

(Kribii et al., 1997), and the complexity of the mammalian

squalene epoxidase reaction which requires, in addition to

NADPH cytochrome P450 reductase, a specific lipid transfer

protein for transfer of squalene to the enzyme (Shibata et al.,

2001). This also suggests that in spite of the differen-

tial induction of the two Medicago SEs in planta, with its

Figure 7. Triterpene and sterol profiles in M. truncatula cell suspension cultures.(a,b) Accumulation of triterpene saponins in response to MeJA. The traces show portions of selective ion chromatograms of extracts from unelicited (a) and 24-hMeJA-elicited (b) M. truncatula cell suspension cultures. 1, rhamnose-hexose-hexose-hederagenin; 2, 3-rhamnose-galactose-glucose-soyasapogenol B; 3,rhamnose-hexose-hexose-soyasapogenol E.(c,d) Effects of MeJA on sterol levels. The traces show portions of selective ion chromatograms of extracts from unelicited (c) and 24-h MeJA-elicited (d)M. truncatula cell suspension cultures. All compounds are detected as the TMS ethers. 1, stigmasterol; 5, [22E]-ergosta-7,22-dien-3a-ol; 6, b-amyrin; 8, stigmata-7,24[28]-dien-33a-ol. Compounds 2, 3, 4 and 7 are unidentified sterols.

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Triterpene saponin biosynthesis 1043

implications for differential function, both forms might be

able to participate in sterol biosynthesis in plant cells, as in

the heterologous yeast system.

The formation of b-amyrin by cyclization of 2,3-oxidos-

qualene is a complex reaction believed to occur via the

‘chair-chair-chair’ conformation of the substrate. The OSCs

lanosterol and cycloartenol synthase have been extensively

studied in mammals and yeast (Abe and Prestwich, 1995;

Corey et al., 1993, 1994, 1996; Morita et al., 1997). P. ginseng

b-AS has been previously cloned and functionally

expressed in yeast (Kushiro et al., 1998). Recently, cDNAs

encoding three proteins from A. thaliana with 49–59% iden-

tity to cycloartenol synthase were functionally expressed.

The products of one of these enzymes consisted of a

mixture of lupeol, b-amyrin, and a-amyrin (15 : 55 : 30) (Hus-

selstein-Muller et al., 2001). This enzyme is distinct from a

lupeol synthase characterized from the same source (Her-

rera et al., 1998). In the present study, M. truncatula b-AS

catalyzed the formation of b-amyrin alone, with no minor

products, as also observed for the Panax enzyme (Kushiro

et al., 1998).

Previous studies have shown effects of sucrose and

mineral nutrients on saponin production in plant cell sus-

pension cultures, but these effects were neither large nor

rapid (Akalezi et al., 1999; Fulcheri et al., 1998). M. truncatula

root cell suspension cultures produce low levels of triter-

pene saponins and have correspondingly low-steady state

levels of SS, SE and, particularly b-AS transcripts. We have

now determined conditions for rapid induction of triterpene

biosynthesis in the cultures following exposure to MeJA.

Jasmonates are important stress signaling molecules

known to be good elicitors for a wide range of secondary

metabolites such as polyamines, coumaryl conjugates,

anthraquinones, naphthoquinones, polysaccharides, terpe-

noids, alkaloids, and phenylpropanoids from different plant

origins (Memelink et al., 2001). In Medicago cell suspension

cultures, exposure to MeJA downregulates the flavonoid

branch of phenylpropanoid biosynthesis, as assessed by

CHS steady state transcript levels, but induces the appear-

ance of glycosides of the triterpenes hederagenin and

soyasapogenols B and E. In contrast, exposure of the cells

to yeast elicitor results in a strong induction of the phenyl-

propanoid pathway associated with accumulation of iso-

flavonoid phytoalexins (H. Suzuki, J.W. Blount, and R.A.

Dixon, unpublished results), but with little effect on triter-

pene biosynthesis. The downregulation of cycloartenol

synthase transcripts following exposure to MeJA suggests

preferential channeling of oxidosqualene from sterol synth-

esis to triterpene synthesis following elicitation, in a similar

manner to the regulation at the sesquiterpene/sterol branch

in elicited tobacco cell cultures (Vogeli and Chappell, 1988).

However, metabolic profile analysis failed to demonstrate

a reduction in sterol levels in MeJA-elicited M. truncatula

cell cultures, at least at the 24-h time point tested. Thus,

although cycloartenol synthase transcript levels are down-

regulated by MeJA treatment, they do not completely dis-

appear but rather reach a new steady state, which may still

be sufficient to support sterol biosynthesis.

MeJA is a wound signal for activation of plant responses

to insect attack (McConn et al., 1997). It is interesting that

high expression of b-AS appears to be associated with

insect herbivory in M. truncatula. There is a correlation

between growth of the yellow mealworm (Tenebrio moli-

tor) and saponin content in M. sativa, with glycosides of me-

dicagenic acid exhibiting the highest degree of antifeedant

activity (Pracros, 1988). Preliminary results indicate that

regurgitant from tobacco hornworm (Korth and Dixon,

1997) can induce b-AS transcripts both in intact plants

(K. Korth, personal communication) and cell cultures

(L. Achnine and H. Suzuki, unpublished results) of M.

truncatula. The cell culture system may therefore prove a

use ful model for the study of anti-insect responses in

legumes.

The later enzymes of triterpene biosynthesis are primarily

cytochrome P450s and glycosyl transferases. These

enzymes exist as large supergene families in plants (Chap-

ple, 1998; Vogt and Jones, 2000), with approximately 250

members of the P450 family estimated from current

M. truncatula EST information. Glycosyltransferase activity

has been shown to correlate with saponin production in

root cultures of Gypsophila paniculata (Herold & Henry,

2001), but specific triterpene glycosyltransfereases remain

to be characterized at the molecular level. The ability to

differentially upregulate two major pathways of natural

product metabolism (i.e. triterpenes and phenylpropa-

noids/flavonoids) in a cell culture system facilitates the

design of DNA macro- and microarray experiments for

selection of candidate P450 and glycosyltransferase genes

from our extensive EST collections (Bell et al., 2001). These

can then be functionally characterized in yeast (P450s)

or E. coli (GTs). It is possible that the cytochrome P450s

might represent important regulatory steps in triterpene

biosynthesis.

Experimental procedures

Plant material

M. truncatula Gaerth ‘Jemalong’ (line A17) plants were grownunder greenhouse conditions in 11 cm diameter pots in Metro-mix 250 or 350 (Scott, Marysville, OH, USA), nine plants per potwith an 18-h light/258C and 6-h dark/228C photoperiod. Cell sus-pension cultures were initiated from roots of line A17, maintainedin a modified Schenk and Hildebrandt medium, as describedpreviously for alfalfa cultures (Dixon et al., 1981), and subculturedevery 10–14 days.

Six days after subculture, dark grown M. truncatula A-17 rootsuspension cultures (75-ml batches) were treated with MeJA(500 mM), YE (50 mg glucose equivalents ml�1), SA (500 mM), orABA (500 mM), harvested at various times after elicitation and

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1044 Hideyuki Suzuki et al.

frozen at �808C. Control cells were treated with the same volumeof distilled water.

Extraction and quantitation of triterpene saponins and

sterols

Saponin extracts were obtained using a solid phase extractionprocedure as previously described (Oleszek, 1988; Oleszek et al.,1990). Six grams fresh weight of cells was extracted in 80%methanol for 24 h. The extracts were concentrated under a nitro-gen stream to yield an aqueous solution that was diluted to a finalconcentration of 35% methanol (v/v) and loaded onto a 35-ml, 10-g,C18 SPE extraction cartridge (Waters, Milford, MS). The SPEcartridge was washed with two column volumes each of HPLCgrade water and 35% methanol. The saponins were eluted withtwo column volumes of 100% methanol. The methanol fractionwas dried under vacuum, re-suspended in methanol to a finalconcentration of c. 400 ng ml�1 and analyzed by gradient elution,reverse-phase HPLC with simultaneous online UV and mass selec-tive detection (Huhman and Sumner, 2002).

For determination of sterol levels, 6.0 mg of lyophilized tissuewas extracted with 1.5 ml water and 1.5 ml chloroform at 508C for2 h. The sample was centrifuged at 2900 g for 30 min and 1.0 ml ofthe organic layer collected and dried under a stream of nitrogen.Lipids were hydrolyzed in 0.8 ml chloroform and 0.5 ml of metha-nol:HCl (97 : 3) for 2 h at 508C. The samples were washed with three0.5-ml aliquots of HPLC grade water to remove acid, dried, re-suspended in 50 ml pyridine, and derivatized with 50 ml MSTFA (N-methyl-N-trimethylsilyltriflouroacetamide) plus 1% TMCS (tri-methylchlorosilane). One microliter of sample was injected usingan HP 6890 series autosampler onto an HP 6890 GC with a DB-5MScolumn (60 m, 0.25-mm film thickness, 0.25 mm i.d., J & W Scien-tific). Spectroscopic data were acquired with an HP 5973 massselective detector scanning from 50 to 650 at a scan rate of 2.48scans sec�1. Best matches were assigned by searching the NISTlibrary following spectral de-convolution by AMDIS software.

Functional expression of M. truncatula SS, SE, and b-AS

Expression of M. truncatula EST clone NF066G09IN, a putativesqualene synthase, was performed by amplification of the openreading frame from pBluescript II SKþ (Stratagene, La Jolla, CA)with introduction of NcoI and BamHI sites (50-CCATGCCATGG-GAAGTATAAAAGCGATTTTGAAGAATC-30 for the upstream pri-mer and 50-CGGGATCCTTAGTTATTGTGACGATTGGCAGAGAG-30

for the downstream primer). The PCR product was purified, ligatedinto pGEMTeasy vector (Promega, Madison, WI, USA), sequenced,excised and re-cloned between the NcoI and BamHI sites of thepET15b expression vector (Novagen, Madison, WI, USA). E. coliBL21 (DE3, pLyS) cells harboring the expression construct weregrown to an OD600 of 0.6, and expression was induced by additionof isopropyl 1-thio-b-D-galactopyranoside (IPTG) to a final con-centration of 0.5 mM, with further incubation for 3 h. Cell lysateswere prepared and the crude extract used for the enzyme assay.The reaction mixture contained, in a total volume of 100 ml, 11.4 mM

[1,2–14C]-FPP (125 nCi; American Radiolabeled Chemicals, St.Louis, MO), 3 mM NADPH, 5 mM MgCl2, 0.1 mM dithiothreitol,100 mM KF, 50 mM Tris–HCl (pH 7.6), and enzyme (70mg of pro-tein). The reaction mixture was incubated at 308C for 1 h and thereaction stopped by addition of 100 ml ethyl acetate. Lipids wereextracted with ethyl acetate and 10 ml subjected to TLC on silica gelreverse phase plates (RP-18) (J.T. Baker, Phillipsburg, NJ). Theplates were developed with acetone: water (19 : 1, v/v). After devel-

opment, plates were exposed and analyzed with a bio-imageanalyzer (Molecular Dynamics, Sunnyvale, CA).

Functional identification of putative squalene epoxidasesencoded by M. truncatula EST clones NF065G06EC (SE1) andNF102D09LF (SE2) was achieved by heterologous expression inthe Erg1 knockout yeast mutant KLN (MATa, erg1::URA3, leu2,ura3, trp) (Landl et al., 1996). The PCR fragments with introducedBamHI and XhoI sites were amplified with the following primers:for the pWV3-SE1 construct, 50-CGCGGATCCATGATAGACCCC-TACGGTTTCGGGTGG-30 for upstream and 50- CCGCTCGAGT-TATGCATCTGGAGGAGCTCTATAAT-30 for downstream; forthe pWV3-D47SE1 construct, 50-CGCGGATCCATGTCTTTTAATCC-CAACGGCGATGTTG-30 for upstream; for the pWV3-SE2 construct,50-CGCGGATCCATGGATCTATACAATATCGGTTGGAATTTA-30 forupstream and 50-CCGCTCGAGTCAAAATGCATTTACCGGGGGAG-CTC-30 for downstream; for the pWV3-D52SE2 construct, 50-CGC-GGATCCATGTCGGACAAACTTAACGGTGATGCTG-30 forupstream.For amplification of the yeast Erg1 sequence, 50-CGGGATC-CATGTCTGCTGTTAACGTTGCACCTGAATTG-30 was used for theupstream primer and 50-CCGCTCGAGTTAACCAATCAACTCAC-CAAACAAAAATGGG-30 for downstream. The PCR products werepurified, subcloned into pGEMTeasy vector, sequenced, excised,and re-cloned between the BamHI and XhoI sites of the pWV3 yeastexpression vector. The SE1 and SE2 ORFs, SE1 with 47 amino acidstruncated from the N-teminus, SE2 with 52 amino acids truncatedfrom the N-teminus, and the Erg1 ORF as a positive control, wereunder control of the constitutive ADH1 promoter, and the pWV3vector contained the Leu2 selectable marker for yeast expression.Anaerobic conditions were achieved by culturing the yeast strainsin an Anaerocult A chamber (VWR Scientific Products, Atlanta,GA). Ergosterol (final concentration 20 mg ml�1) was dissolved inTween 80:ethanol (1 : 1, v/v), with a final Tween 80 concentration of0.5% (v/v) in the medium. EST clone NF051E06IN contained anapparent full-length oxidosqualene cyclase (OSC) gene in pBlue-script SK– vector. The plasmid was digested with NotI, XhoI, andScaI, to release the 2.8-kb insert with NotI and XhoI termini (ScaIwas included to cut the 2.9-kb vector into 1.1- and 1.8-kb fragments,facilitating purification). The insert was subcloned into the yeastexpression vector pRS426GalR that contains the URA3 selectablemarker, the 2m origin of replication, and Gal promoter. This high-copy expression construct was named pRX10.2, and was trans-formed into yeast lanosterol synthase mutant SMY8 (MATaerg7::HIS3 hem1::TRP1 ura3–52 trp1-D63 leu2–3112 his3-D200ade2 Galþ). The transformants were selected on synthetic com-plete medium (containing 2% dextrose) lacking uracil and supple-mented with heme (13 mg ml�1), ergosterol (20 mg ml�1), andTween-80 (5 ml ml�1). SMY8 harboring empty vector pRS426Galwas used as negative control in the following assay.

A 5-ml yeast culture was induced with 2% (w/v) galactose andgrown to saturation. The harvested yeast cells were re-suspendedin 200 mM sodium phosphate buffer (pH 6.4), lysed by vortexingwith glass beads, and incubated with 1 mg ml�1 oxidosqualeneand 0.1% Tween-80. The reaction was incubated at room tempera-ture for 24 h and quenched with two volumes of ethanol. Aftercentrifugation, the supernatant was transferred into a glass tube,and the cell debris was extracted with two further volumes ofethanol. The combined ethanol extract was dried under a nitrogenstream, re-dissolved in ethyl acetate and filtered through a smallsilica gel plug to remove cell debris and some polar components.The crude extract was derivatized to form trimethylsilyl (TMS)ethers by treatment with 50 ml of bis(trimethylsilyl)trifluoroaceta-mide-pyridine (1 : 1, v/v) at 408C for 2 h and was analyzed by GC-FIDand GC–MS, with epicoprostanol (an unnatural C-30 sterol) TMSether as internal standard and authentic b-amyrin TMS ether as

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Triterpene saponin biosynthesis 1045

external standard. Co-injection of crude product(s) and b-amyrinstandard was performed on GC–MS.

GC analysis employed a Hewlett-Packard 6890 system equippedwith a Rtx-5 capillary column (Restek, 30 m� 0.25 mm i.d., 0.10 mmdf). A 5-ml aliquot was injected at 2808C with a split ratio of 40 : 1,helium flow was at 20 cm sec�1, and the following temperatureprogram was applied: 1008C for 2 min, rising to 2808C at208C min�1, holding at 2808C for 30 min. The flame ionizationdetector was at 2808C. GC–MS was performed on a Hewlett-Pack-ard 5890 A instrument equipped with a DB-5ms column (J & W, 60m� 0.25 mm i.d., 0.10 mm df). Separation was achieved with split-less injection (1-min delay) at 2008C, helium flow at 30 cm sec�1

(1 ml min�1) and the identical temperature program as above.Mass spectra (m/z 35–500) were obtained on a ZAB-HF reverse-geometry double-focusing instrument at 70 eV with an electron-impact ion source (2008C). The accelerating voltage was 8 kV andthe resolution was 1000 (10% valley).

A 300-ml SMY8[RX10.2] yeast culture was processed similarly toobtain enough enzymatic product(s) for NMR analysis. The etha-nolic supernatant of the in vitro catalytic reaction was evaporatedto dryness and re-dissolved in ethyl acetate. The crude mixturewas filtered through a silica plug and then separated by silica gelcolumn chromatography to remove excess oxidosqualene sub-strate, exogenous ergosterol, and fatty acids. Polycyclic triterpenealcohols co-migrate with b-amyrin on TLC, and fractions withmaterial in this region were pooled and analyzed by 1H-NMRand GC–MS, which showed b-amyrin uncontaminated by othertriterpene alcohol isomers (detection limit 2%). NMR spectra offree sterols were obtained on a Bruker AMX500 spectrometer(500 MHz for 1H) at 258C in CDCl3 solution and referenced tointernal tetramethylsilane.

DNA and RNA gel blot analysis

DNA was extracted from M. truncatula plants by standard methods(Sambrook et al., 1989). Southern blotting and hybridization werecarried out as described previously (Church and Gilbert, 1984). TheSS, SE1, SE2, and b-AS probes were amplified as complete ORFsfrom the EST clones NF066G09IN, NF065G06EC (SE1),NF102D09LF (SE2), and NF051E06IN, respectively. For generationof gene-specific probes to distinguish between the SE1 and SE2transcripts, pBluescriptSE1 was double digested with EcoRI andNaeI, and pBluescriptSE2 was digested with EcoRI and AatII,resulting, respectively, in 195- and 247-bp fragments specific forthe N-termini of the two open reading frames.

Thirty micrograms of M. truncatula RNA from root cell suspen-sion culture or other tissues was separated by electrophoresis in a1% agarose gel containing 0.66 M formaldehyde and then blottedonto a Hybond-Nþ membrane (Amersham). The gene-specific SE1and SE2 probes and the entire cDNA fragments of SS, b-AS, andthe M. truncatula cycloartenol synthase (NF015H10LF), phenylala-nine ammonia lyase (NF011C12ST), and chalcone synthase(NF044D07EC) EST clones were radiolabeled with [32P] dCTP usinga Ready-to-go DNA Labeling Beads (-dCTP) kit (Amersham) andused as probes.

Acknowledgements

The authors thank Drs R. Leber and F. Turnowsky (Institute ofMolecular Biology, Graz University, Austria) for providing theKLN1 yeast strain, Dr Wayne Versaw (Noble Foundation) for thepWV3 yeast expression vector, Angela Scott for EST sequencing,David Huhman for LC/MS analysis of saponins, Corey Broeckling

and Jihai Pang for GC/MS analysis of sterols and b-amyrin, and DrsLiangjiang Wang and Robert Gonzales for critical reading of themanuscript. This work was supported by the Samuel RobertsNoble Foundation and in part by grants to R.A.D. from the NationalScience Foundation (0109732) and to S.P.T.M. from the USDA(2001-35315-10157) and the Robert A. Welch Foundation (C-1323).

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