Biosynthetic diversity in plant triterpene cyclizationDereth R Phillips, Jeanne M Rasbery, Bonnie Bartel and Seiichi PT Matsuda

Plants produce a wealth of terpenoids, many of which have

been the tools of healers and chefs for millennia. Recent

research has led to the identification and characterization of

many genes that are responsible for the biosynthesis of

triterpenoids. Cyclases that generate sterol precursors can be

recognized with some confidence on the basis of sequence;

several catalytically important residues are now known, and the

product profiles of sterol-generating cyclases typically reflect

their phylogenetic position. By contrast, the phylogenetic

relationships of cyclases that generate nonsteroidal triterpene

alcohols do not consistently reflect their catalytic properties

and might indicate recent and rapid catalytic evolution.

Addresses

Department of Biochemistry and Cell Biology and Department of

Chemistry, Rice University, 6100 Main Street, Houston, TX 77005, USA

Corresponding author: Matsuda, Seiichi PT (matsuda@rice.edu)

Current Opinion in Plant Biology 2006, 9:305–314

This review comes from a themed issue on

Physiology and metabolism

Edited by Eran Pichersky and Krishna Niyogi

Available online 3rd April 2006

1369-5266/$ – see front matter

# 2005 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.pbi.2006.03.004

IntroductionPlants have long been known to synthesize a plethora of

small molecules, and plant genome sequencing is reinfor-

cing the view that plants devote considerable efforts

towards developing chemical solutions to biological prob-

lems. Terpenoids are metabolites of isopentenyl pyro-

phosphate (IPP) oligomers and comprise the largest group

of plant natural products, with over 20 000 known mem-

bers. Triterpenoids are synthesized from IPP via the 30-

carbon intermediate squalene, and include sterols, ster-

oids, and triterpenoid saponins (Figure 1). In addition to

sterols (which are 6,6,6,5-tetracycles derived from lanos-

terol or cycloartenol), nearly 100 additional triterpenoid

skeletons have been described [1��]. Cycloartenol and

lanosterol can serve as precursors to membrane sterols and

steroid hormones. Other triterpenes have less well-

defined roles, but many might act in plant defense.

Triterpenoid carbon frameworks are cyclized by mem-

bers of the oxidosqualene cyclase (OSC) family, which

has expanded greatly in plants. In this review, we

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highlight recent progress in the study of the enzyme

families that generate triterpene skeletal diversity.

Oxidosqualene cyclases that utilize theprotosteryl cationOxidosqualene cyclases (OSCs) convert oxidosqualene to

one or more cyclic triterpene alcohols with up to six

carbocyclic rings (Figure 1). Plants biosynthesize diverse

triterpenoids and encode multiple OSC enzymes to form

these skeletons (Table 1). For example, the sequenced

genomes of Arabidopsis thaliana and Oryza sativa (rice)

encode 13 [1��,2] and nine apparent OSC enzymes,

respectively. Genome mining, heterologous expression,

and biochemical characterization of the encoded proteins

(Table 1) have provided substantial insight into the

triterpenoid biosynthetic capabilities of plants.

Cycloartenol synthase (CAS) converts oxidosqualene to

cycloartenol through the protosteryl cation intermediate

(Figure 1) and was the basal plant OSC from which others

derived [3��]. The Arabidopsis CAS1 cDNA was cloned by

screening extracts from a yeast lanosterol synthase mutant

(erg7) that was transformed with random ArabidopsiscDNAs for the ability to cyclize oxidosqualene [4]. This

isolation facilitated the subsequent homology-based iden-

tification of other OSCs. Plant CAS genes have now been

cloned and characterized from numerous eudicots, several

monocots, and a gymnosperm (Table 1, Figure 2), consis-

tent with biochemical evidence of CAS activity throughout

seed plants [5–7]. The similarity of plant CAS genes to

those in amoebae [8] and bacteria [9] suggests that the

known cycloartenol synthases are orthologs, and that

cycloartenol synthase predates the emergence of plants.

Lanosterol is the initial carbocyclic sterol precursor in

animals, fungi, and trypanosomatids. Although substan-

tial labeling experiments support cycloartenol rather than

lanosterol as the major plant sterol precursor [5,10],

lanosterol biosynthesis has been demonstrated in a few

plants (e.g. in the latex of several Euphorbia species [11]).

Intriguingly, the Arabidopsis protein most similar to CAS1

(At3g45130, 65% identical) encodes a lanosterol synthase

(LSS) [12��]. The presence of probable At3g45130/LSS

orthologs in the asterids Taraxacum officinale and Panaxginseng and the eurosid Luffa cylindrica (66–69% identical;

Figure 2) suggests that lanosterol synthase is broadly

distributed among eudicots. The maintenance of CAS

in all examined plant lineages (Table 1), despite an

apparent ability of at least some plants to produce lanos-

terol [12��], implies that plants require some cycloartenol

metabolites that cannot be made from lanosterol. Muta-

tional analyses have shown that Tyr410, His477, and

Current Opinion in Plant Biology 2006, 9:305–314

306 Physiology and metabolism

Figure 1

A simplified scheme of plant triterpenoid biosynthesis. Farnesyl diphosphate synthase (FPS) isomerizes isopentenyl diphosphate (IPP) and

dimethylallyl diphosphate (DMAPP) to farnesyl diphosphate (FPP), which squalene synthase (SQS) converts to squalene. Squalene epoxidase

(SQE) oxidizes squalene to 2,3-oxidosqualene. OSC enzymes cyclize 2,3-oxidosqualene through cationic intermediates (e.g. the protosteryl cation,

the dammarenyl cation and others not shown) to triterpene alcohols or aldehydes. A few of the characterized OSC enzymes and products

listed in Table 1 are illustrated. OSC products can be further modified by multiple enzymes to form membrane sterols, brassinosteroids, saponins,

and other compounds. bAS1, b-amyrin synthase; LUP, lupeol synthase; MRN1, marneral synthase; PP, diphosphate; THA1, thalianol synthase.

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Biosynthetic diversity in plant triterpene cyclization Phillips et al. 307

Table 1

Characterized plant oxidosqualene cyclases.

Synthase Plant Name (Genbank accession)a Productsb (product identification method [reference[s]])

Cycloartenol Abies magnifica CAS1 (AAG44096) Cycloartenol (NMR, GC;c)

Cycloartenol A. thaliana At2g07050/CAS1 (P38605) Cycloartenol (NMR, IR, MS [4])

Cycloartenol B. platyphylla BPX1 (BAB83085)

BPX2 (BAB83086)

Cycloartenol (LC-MS [53])

Cycloartenol C. speciosus CSOSC1 (BAB83253) Cycloartenol (LC-MS [38])

Cycloartenol C. pepo CPX (BAD34644) Cycloartenol (LC-MS [17��])

Cycloartenol G. glabra GgCAS1 (BAA76902) Cycloartenol [54]

Cycloartenol L. cylindrica LcCAS1 (BAA85266) Cycloartenol [55]

Cycloartenol P. ginseng PNX (BAA33460) Cycloartenol (HPLC [56])

Cycloartenol P. sativum CASPEA (BAA23533) Cycloartenol [57]

Lanosterol A. thaliana At3g45130/LSS1/PEN7 Lanosterol (GC-MS, NMR [12��])

Cucurbitadienol C. pepo CPQ (BAD34645) Cucurbitadienol (LC-MS, NMR [17��])

b-Amyrin Avena strigosa AsbAS1 (CAC84558) b-Amyrin (Genetic analysis, HPLC [19])

b-Amyrin B. platyphylla BPY (BAB83088) b-Amyrin (LC-MS [53])

b-Amyrin Euphorbia tirucalli EtAS (BAE43642) b-Amyrin (GC-MS, NMR [58])

b-Amyrin G. glabra GgbAS1 (BAA89815) b-Amyrin (LC-MS [59])

b-Amyrin M. truncatula MtAMY1/bAS1 (CAD23247) b-Amyrin (GC-MS, NMR [23]; HPLC [24])

b-Amyrin P. ginseng PNY1 (BAA33461)

PNY2 (BAA33722)

b-Amyrin (HPLC, MS, NMR [56,60])

b-Amyrin P. sativum PSY (BAA97558) b-Amyrin (LC-MS, GC-MS, NMR [32])

Lupeol B. platyphylla BPW (BAB83087) Lupeol (LC-MS [53])

Lupeol G. glabra GgLUS1 (BAD08587) Lupeol (LC-MS [26��])

Lupeol O. europaea OEW (BAA86930) Lupeol (LC-MS, NMR [29])

Lupeol T. officinale TRW (BAA86932) Lupeol (LC-MS, NMR [29])

Thalianol A. thaliana At5g48010/THA1/PEN4 Thalianol (NMR [39��])

Marneral A. thaliana At5g42600/MRN1/PEN5 Marneral (NMR [40��])

Isomultiflorenol L. cylindrica LcIMS1 (BAB68529) Isomultiflorenol (LC-MS [30])

Mixed A. thaliana At1g78970/LUP1 Lupeol, 3b,20-dihydroxylupane, b-amyrin, germanicol, taraxasterol

and c-taraxasterol (GC, MS [33]; NMR [33–35])

Mixed A. thaliana At1g78960/LUP2/

YUP8H12R.43

b-Amyrin, taraxasterol, tirucalla-7,21-dien-3b-ol, lupeol, bauerenol,

butyrospermol, multiflorenol, a-amyrin and c-taraxasterol

(HPLC, NMR [2,36]; GC-MS [2]; LC-MS [36])

Mixed A. thaliana At1g66960/LUP5 Tirucalla-7,21-dien-3b-ol and additional uncharacterized (LC-MS [37])

Mixed A. thaliana At1g78500/PEN6 Bauerenol, lupeol, a-amyrin and additional uncharacterized (LC-MS [37])

Mixed C. speciosus CsOSC2 (BAB83254) Lupeol, germanicol, b-amyrin and additional uncharacterized (LC-MS [38])

Mixed L. japonicus AMY2 (AAO33580) Lupeol, b-amyrin and additional uncharacterized (HPLC [24])

Mixed P. sativum PSM (BAA97559) a-Amyrin, b-amyrin, d-amyrin, c- taraxasterol, butyrospermol,

lupeol, germanicol and taraxasterol (LC-MS, GC-MS, NMR [32])

a A. thaliana PEN and LUP designations are from [2].b For OSC enzymes with mixed products, major products are listed in bold text if known.c JBR Herrera, PhD thesis, Rice University, 1999. Abbreviations: GC, gas chromatography; HPLC, high pressure LC; IR, infrared spectroscopy; LC,

liquid chromatography; MS, mass spectrometry; NMR, nuclear magnetic resonance.

Ile481 cooperate to control product specificity in Arabi-dopsis CAS1 [13–15]. In fact, Arabidopsis CAS1 can be

converted to an accurate lanosterol synthase with only

two amino-acid substitutions: His477 to Asn and Ile481 to

Val [16��]. It is intriguing that Arabidopsis LSS contains

residues that correspond to these mutations. This pre-

cedent suggests that only a small evolutionary step could

convert CAS to lanosterol synthase if natural selection

favored a lanosterol route in plants.

Other enzymes that form the protosteryl cation have arisen

from CAS gene duplication and diversification. For exam-

ple, Cucurbita pepo cucurbitadienol synthase (CPQ) is

closely related (65–71% identical) to both cycloartenol

and lanosterol synthases (Figure 2) but produces cucurbi-

tadienol ([17��]; Figure 1), a precursor of cucurbitacins,

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which are bitter compounds that might act as antifeedants

(reviewed in [18]). The sequence similarity of plant

cycloartenol, lanosterol, and cucurbitadienol synthases

(Figure 2) is consistent with relatively recent divergence

of lanosterol and cucurbitadienol synthases from an ances-

tral CAS via an evolutionary route that maintained the

protosteryl cation intermediate (Figure 1).

Cyclases that generate single triterpenesthrough the dammarenyl cationSeveral oxidosqualene cyclases that function in secondary

metabolism also have been reported to generate single

products. Lupeol synthases cyclize oxidosqualene to the

dammarenyl cation, promote ring expansion and annula-

tion to the lupyl cation, and terminate by abstracting the

C-29 proton to form lupeol (Figure 1; [1��]). Lupeol

Current Opinion in Plant Biology 2006, 9:305–314

308 Physiology and metabolism

Figure 2

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Biosynthetic diversity in plant triterpene cyclization Phillips et al. 309

synthases are found in Glycyrrhiza glabra, Betula platy-phylla, T. officinale, and Olea europea (Table 1); these

enzymes are 74–81% identical to one another and form

a clade that is distinct from other characterized OSCs

(Figure 2). The presence of eurosid and asterid genes in

this clade suggests that an accurate lupeol synthase

evolved before the divergence of asterids and eurosids.

b-Amyrin synthases also form the lupyl cation, but allow

further ring expansion and some rearrangement before

deprotonation to b-amyrin [1��]. Several OSCs from

eudicots and monocots produce b-amyrin accurately

(Table 1). These b-amyrin synthases are considerably

more distant from one another (48–50% identical) than

are the CAS enzymes (70–79% identical), and indepen-

dent origins of b-amyrin synthases in eudicots and mono-

cots have been proposed [19,20��].

Lupeol, b-amyrin, and their diverse metabolites are

implicated in various plant processes. b-Amyrin is a

precursor of saponins, which are triterpene glycosides

such as the antifungal saponin avenacin found in Avenaroots [19,20��,21]. b-Amyrin and its metabolites tend to

accumulate in specific tissues (see [22��] for the localiza-

tion of 31 Medicago saponins). Some of this localization

could be transcriptional, as indicated by the tissue-spe-

cific expression of b-amyrin synthase genes in Medicago[23,24], Lotus [24], Pisum [24], Centella [25], Glycyrrhiza[26��], and Avena [19,20��]. Lupeol synthase expression is

associated with root nodulation in several plants. For

example, Glycyrrhiza lupeol synthase is expressed in

nodules and cultured cells, the same areas in which the

lupeol metabolite betulinic acid is detected [26��]. In

Vicia faba nodule outer cortex, 82% of the organic-soluble

material is betulin, a lupeol metabolite, and another 7% is

lupeol [27]. Moreover, a partial OSC sequence

(CAA75588), which was recovered in a screen for genes

that are induced during Medicago truncatula nodulation

[28], is closely related to lupeol synthases [24,29] and is

expressed most highly in root nodules [24]. The role of

lupeol in root nodule biology remains to be elucidated.

In addition to lupeol and b-amyrin synthases, less broadly

distributed enzymes can also have high product

(Figure 2 Legend) Predicted phylogenetic relationships among characterize

enzymes (colored) from Table 1 and uncharacterized enzymes (in black) we

using the Clustal W method. The untrimmed alignment was used to generate

using PAUP 4.05b [52]. The bootstrap method was performed for 100 replic

criterion. Bootstrap values are listed at nodes. Characterized enzymes and c

products are enclosed in similarly colored boxes. GenBank accession numb

CAC84559; Avena ventricosa CS1, AAT38892; Centella asiatica OSCCCS, A

ABB76767; Allium macrostemon ALLOSC1, BAA84603; Taraxacum officinal

LcOSC2, BAA85267; Cucurbita pepo CPR, BAD34646; Avena ventricosa bA

CabAS, AAS01523; Aster sedifolius OXA1, AAX14716; Ricinus communis L

enzymes are indicated by ’Arabidopsis’ followed by the gene locus number

of the respective colored box. Mixed dammarenyl compounds are labeled ‘

according to the cation intermediate through which they create their produc

inconsistent with the overall cation-intermediate type of the clade.

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selectivity. Luffa cylindrica isomultiflorenol synthase

(LcIMS1) cyclizes oxidosqualene through a dammarenyl

cation to make isomultiflorenol [30], a bryonolic acid

precursor [31]. Temporal expression studies in L. cylin-drica cell cultures indicate that LcIMS mRNA levels

correlate with bryonolic acid accumulation [30].

Multifunctional cyclases that generatetriterpenes through the dammarenyl cationThe diversity of plant triterpenes emanates not only from

the radiation of the OSC gene family but also from the

ability of some family members to contribute multiple

products (Table 1). Several subfamilies of multifunc-

tional triterpene synthases use dammarenyl cation inter-

mediates. Orthologous mixed-amyrin synthases have

been characterized from the legumes Pisum sativum(i.e. PSM) [32] and Lotus japonicus (i.e. AMY2) [24].

Arabidopsis encodes several paralogous multifunctional

enzymes that biosynthesize b-amyrin, lupeol, taraxas-

terol, and/or related dammarenyl-derived compounds

(Table 1): At1g78970/LUP1 [33–35], At1g78960/LUP2

[2,36], At1g66960/LUP5 [37], and At1g78500/PEN6 [37].

The legume mixed-amyrin synthases share a recent

common ancestor with accurate b-amyrin synthases;

the Arabidopsis mixed-amyrin synthases are more distant

(Figure 2).

Multifunctional OSC enzymes have arisen repeatedly

during angiosperm evolution (Figure 2). A distant exam-

ple is the Costus speciosus CsOSC2, which makes lupeol,

germanicol, and b-amyrin [38] from the dammarenyl

cation. Rather surprisingly, its closest relatives are

cycloartenol synthases (67–70% identical), which use

the protosteryl cation intermediate (Figure 2). This rela-

tionship implies a recent evolutionary transition that

involved a dramatic catalytic change from the protosteryl

cation to the dammarenyl cation [3��].

Discovery of new triterpene skeletons bygenome miningGenome mining in Arabidopsis has uncovered two

enzymes that generate incompletely cyclized structures

that have not been characterized by classical natural

product isolation. {At5g48010}/THA1 [39] converts

d and predicted plant oxidosqualene cyclases. Characterized

re aligned with the MegAlign program (DNASTAR, Inc., Madison, WI)

a phylogram rooted with Dictyostelium discoideum CAS1 (AAF80384) [8]

ates with all characters weighted equally and distance as the optimality

losely related uncharacterized enzymes that are predicted to have similar

ers for uncharacterized enzymes are as follows: Avena strigosa CS1,

AS01524; Oryza sativa CAS1, AAF03375; Ricinus communis CAS,

e TRV, BAA86933; Panax ginseng PNZ1, BAA33462; Luffa cylindrica

S1, AAT38898; Panax ginseng bAS1, BAD15332; Centella asiatica

UP, ABB76766; and Euphorbia tirucalli EtOSC, BAE43643. A. thaliana

. The product(s) made by each enzyme type are listed to the right

mixed’. The two plant clades separated with a dashed line are labeled

ts (protosteryl or dammarenyl). Asterisks mark products that are

Current Opinion in Plant Biology 2006, 9:305–314

310 Physiology and metabolism

Figure 3

Predicted phylogenetic relationships among enzymatically characterized

and putative squalene epoxidases from sequenced plant genomes.

Sequences are from M. truncatula (Medicago SE1, CAD23249 and

Medicago SE2, AD23248), O. sativa (Oryza 1, Os03g12900 and Oryza 2,

Os03g12910), A. thaliana (At1g58440, At2g22830, At4g37760,

At5g24140, At5g24150 and At5g24160), and Populus trichocarpa

(Populus 1, eugene3.04180003; Populus 2, estExt_Genewise1_v1.C

LG II0395; Populus 3, estExt_fgenesh1_pg v1.C LG V0617; Populus 4,

fgenesh1_pg.C LG XIX000144) accessed through the US Department

of Energy (DOE) Joint Genome Institute website (http://genome.jgi-

psf.org/Poptr1/Poptr1.home.html). They were aligned with the MegAlign

program (DNASTAR, Inc., Madison, WI) using the Clustal W method.

Amino-acid residues that correspond to positions 60–522 from

At1g58440 were used to generate a phylogram that is rooted with

Saccharomyces ERG1 (CAA97201) using PAUP 4.05b [52]. The

bootstrap method was performed for 1000 replicates with all characters

weighted equally and distance as the optimality criterion. Boostrap

values are listed at nodes. Characterized enzymes are in bold font.

The shaded region outlines enzymes that seem most likely to encode

genuine squalene epoxidases on the basis of their similarity to the

characterized Medicago enzymes.

oxidosqualene to the tricyclic alcohol thalianol (Figure 1).

Triterpenoids that have the methyl substitution and

olefinic position of thalianol have not been found in

nature. At5g42600/MRN1 [40��] catalyzes an unusual

cyclization reaction: oxidosqualene is converted to a

bicyclic cation that undergoes rearrangement and A-ring

cleavage to generate a monocyclic aldehyde (Figure 1).

Marneral also has not been isolated from any natural

source but is suggested as a biosynthetic precursor of

iridals found in sword lilies [41]; these monocots are only

distantly related to the eudicot Arabidopsis. These exam-

ples demonstrate that heterologous expression of novel

OSC enzymes can provide a means to mine plant gen-

omes for new biosynthetic pathways and low-abundance

natural products.

Squalene epoxidasesOSC enzymes are a major radiation point in the triterpe-

noid pathway and facilitate the production of numerous

and diverse triterpenoids. Interestingly, plants also have

multiple genes that are predicted to encode squalene

epoxidase (SQE) enzymes, also known as squalene mono-

oxygenases. SQEs catalyze the first oxygenation step in

triterpene biosynthesis, converting squalene into the

OSC precursor 2,3-oxidosqualene (Figure 1).

The first cloned SQE gene, from Saccharomyces cerevisiae,was identified by complementation of the yeast ergosterol

biosynthetic mutant erg1 [42]. Two Medicago squalene

epoxidases that were identified by sequence similarity are

able to rescue the yeast erg1 mutant defect when hetero-

logously expressed [23], indicating that the encoded

enzymes function as squalene epoxidases.

In addition to the two biochemically characterized Med-icago SQEs [23], the sequenced genomes of Oryza, Popu-lus, and Arabidopsis (Figure 3) encode multiple predicted

SQEs. Each of these plants possesses genes that are

closely related (63–82% identical) to the characterized

Medicago SQEs, but Arabidopsis also has three SQE-like

genes (At5g24140, At5g24150 and At5g24160) that are

more diverged (44–48% identical, Figure 3). It remains to

be determined which of these more distant enzymes are

genuine SQEs and whether any have adopted new cat-

alytic functions.

Functional implications of triterpenediversificationSeveral forces could drive expansion in the SQE and OSC

families. Phylogenic analyses of Arabidopsis and Oryzareceptor-like kinases reveal that the expansion of a parti-

cular sub-family after speciation can indicate that mem-

bers have evolved to act in defense or disease resistance

[43��]. Conversely, very little post-speciation expansion is

observed when sub-family members function in devel-

opment or central metabolism [43��]. This observation

might begin to explain the divergence patterns within the

Current Opinion in Plant Biology 2006, 9:305–314

plant OSC family. Minimal post-speciation duplication is

observed in the CAS subfamily (Figure 2), and CAS

enzymes from the eudicot Arabidopsis, the monocot C.speciosus, and the gymnosperm Abies have remained very

similar to one another (�75% identical). These observa-

tions are consistent with a housekeeping function for

cycloartenol as a precursor to bulk membrane sterols

and brassinosteroids. By contrast, substantial lineage-

specific expansion is apparent in plant OSC sub-families

that do not use the protosteryl cation intermediate

(Figure 2), consistent with known and postulated defense

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Biosynthetic diversity in plant triterpene cyclization Phillips et al. 311

roles for many non-steroidal triterpenes and suggesting

that plants derive advantage from synthesizing diverse

triterpene skeletons. Although the neighboring positions

of Arabidopsis LUP1 and LUP2 (80% identical) on chro-

mosome 1 imply recent duplication, the product profiles

of these multifunctional OSCs have already diverged

(Table 1).

Why some OSCs produce multiple products is not known

but some of these products might represent evolution in

progress. Numerous mutagenesis and directed evolution

experiments demonstrate that small changes in OSC

sequences can expand product diversity (reviewed in

[44]). If a new product confers an advantage, subsequent

mutations might enhance the production of the beneficial

product. Interestingly, Arabidopsis contains two subfami-

lies of enzymes for which orthologs from other plant

species have not been identified (Figure 2). Of the

characterized enzymes in these subfamilies, four are

multifunctional and two make compounds not yet found

in plants (Table 1).

Control of triterpene metabolismGene duplication, in addition to allowing for product

diversification, allows the differential expression of family

members in various plant tissues. For example, Arabidop-sis maintains two genes that encode farnesyl diphosphate

synthase (FPS), which acts early in terpene biosynthesis

(Figure 1). Arabidopsis FPS1 is expressed in most tissues,

whereas FPS2 expression is restricted to specific devel-

opmental stages [45]. Triterpene biosynthetic genes are

induced to produce terpenoids under specific conditions,

including various biotic stresses. For example, b-amyrin

synthases are induced by a yeast elicitor in Tabernaemon-tana divaricata [46] and M. truncatula [23] cultures, con-

sistent with a role for b-amyrin as a precursor to antifungal

saponins (Figure 1). In addition, treatment with methyl

jasmonate (MeJA), a phytohormone that is involved in

plant stress responses [47], increases the message levels of

confirmed and predicted b-amyrin synthase genes from

Medicago [23], Glycyrrhiza [26��], and Centella asiatica [25]

and is accompanied by increased production of triterpene

saponins [23,25,26��].

In some cases, genetic linkage could provide an efficient

mechanism for the transcriptional control of triterpenoid

biosynthetic genes. In Avena, a cluster of five loci that are

involved in avenacin biosynthesis [20��] is encoded near

the AsbAS1 b-amyrin synthase gene [19]. It will be

interesting to learn whether such clustering is found

for additional plant triterpene metabolic genes.

Metabolic channeling, in which biosynthetic enzymes are

co-localized within a cellular organelle or specific plant

organ, might contribute to pathway control. Substrate

channeling could promote efficient triterpenoid produc-

tion by protecting intermediates from seizure by other

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enzymes that make different products. Channeling might

provide a mechanism to isolate potentially toxic metabo-

lites within particular organelles or might confer benefits

to plants that are challenged with infectious agents or

changing environmental conditions (reviewed in [48��]).

Metabolic channeling may be facilitated by coordinate

expression of genes that encode different steps of a

pathway. For example, when Medicago cell cultures are

treated with MeJA, one squalene epoxidase gene (SE2) is

dramatically upregulated [23]. Genes encoding squalene

synthase and b-amyrin synthase, which precede and

follow SE2 in saponin production, are similarly MeJA-

induced, but SE1 and CAS are not [23]. This coordinate

regulation might indicate separate metabolic channels for

saponin and sterol production in this organism.

Conclusions and future prospectsThrough genome sequencing, heterologous expression,

enzymology, and analytical chemistry, a growing number

of plant triterpenes are being associated with the enzymes

that produce them. Although relatively few OSC

enzymes have been catalytically characterized

(Table 1), progress in this area is rapidly accelerating.

Most characterized plant OSCs are from angiosperms

(Table 1); future OSC characterizations from more basal

land plants would clarify orthologous relationships in the

family. As the number of sequenced and characterized

OSC and SQE enzymes increases, so will our under-

standing of the relationships between various enzyme

subfamilies, which in turn will allow more accurate pre-

dictions of the products of uncharacterized synthases and

more facile metabolic engineering for pharmaceutical or

agricultural benefit.

Compounding the challenge of completing triterpenoid

accounting is the complexity of the metabolism that can

follow oxidosqualene cyclization (reviewed in [49]). Elab-

orations of the triterpene skeleton can include hydrox-

ylation by P450-dependent monooxygenases, alkylation

by methyltransferases, and glycosylation by glycosyltrans-

ferases [50��]; these and other modifications can be

essential for bioactivity. For example, more than 20

enzymatic steps are needed to convert cycloartenol into

the active hormone brassinolide [49]. Although progress

has been made in a few pathways, many of the specific

enzymes that carry out these reactions remain to be

characterized.

The model plant A. thaliana was not known as a source of

novel triterpenes, but the study of its OSCs provided

several interesting observations. In addition to the first

cycloartenol synthase [4], Arabidopsis provided the first

characterized lanosterol synthase from the plant kingdom

[12��], and sequence comparisons suggest that this func-

tion might be shared by additional eudicots (Figure 2).

The characterization of the Arabidopsis thalianol [39��]

Current Opinion in Plant Biology 2006, 9:305–314

312 Physiology and metabolism

and marneral [40��] synthases led to the discovery of

compounds not yet found in nature, implying that our

list of known triterpene skeletons is far from complete

and demonstrating a new means of triterpene discovery.

The Arabidopsis LUP1 enzyme provided the first example

of a multifunctional OSC [33–35], and multifunctionality

is now established as a recurring theme in the OSC family

(Table 1; Figure 2). It is likely that additional insights will

arise from the characterization of the remaining five

Arabidopsis OSC family members (Figure 2).

Whereas the biological roles of sterols and brassinoster-

oids are well known (reviewed in [51]), the functions of

other triterpenoid skeletons are only beginning to be

elucidated. It will be informative to continue the exam-

ination of the expression patterns of triterpene biosyn-

thetic genes and proteins in the relevant plants during

development and in response to biotic and abiotic chal-

lenges. Moreover, phenotypic analysis of plants that lack

or overexpress particular triterpene biosynthetic genes

will be necessary to fully understand triterpene function.

An Avena mutant screen yielded eight loci that were

defective in saponin accumulation [21]; one of these is

defective in a b-amyrin synthase gene and is the only

published example of a plant OSC mutant [19]. The

saponin-deficient oat mutants are susceptible to infection

by Gaeumannomyces graminis, providing a biological role

for b-amyrin in fungal disease resistance [21]. Genetic

analyses of the roles of certain triterpenes in Arabidopsiswill probably be complicated by the genetic linkage of

some OSC genes and the partial redundancy of multi-

functional enzymes that synthesize both b-amyrin and

lupeol, among other products (Table 1). Nevertheless,

available evidence suggests that straightforward mutant

analysis might reveal the biological consequences of, for

example, losing cycloartenol or lanosterol synthase. The

remarkable diversity of plant triterpene products uncov-

ered to date suggests that a corresponding wealth of

triterpene function awaits discovery.

AcknowledgementsWe thank Diana Dugas, Mariya Kolesnikova, William K Wilson, AndrewWoodward, and Quanbo Xiong for comments on the manuscript andapologize to colleagues we were unable to cite due to space limitations.The authors’ triterpene research is supported by the US National ScienceFoundation (NSF) (MCB-0209769) and the Robert A Welch Foundation(C-1323 and C-1309). JMR was supported in part by a training grant fromthe National Institutes of Health (T32-GM08362).

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