Triterpene Biosynthesis in Plants

PP65CH08-Osbourn ARI 8 April 2014 21:43

Ramesha Thimmappa,1 Katrin Geisler,1,∗ Thomas Louveau,1 Paul O’Maille,1,2 and Anne Osbourn1

1Department of Metabolic Biology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom; email: [email protected] 2Food and Health Program, Institute of Food Research, Norwich Research Park, Norwich NR4 7UH, United Kingdom

Annu. Rev. Plant Biol. 2014. 65:225–57 Keywords First published online as a Review in Advance on oxidosqualene cyclases, metabolic gene clusters, specialized metabolites, January 29, 2014 sterols, synthetic biology The Annual Review of Plant Biology is online at plant.annualreviews.org Abstract Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org This article’s doi: The triterpenes are one of the most numerous and diverse groups of plant 10.1146/annurev-arplant-050312-120229 natural products. They are complex molecules that are, for the most part, be- Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. Copyright c 2014 by Annual Reviews. yond the reach of chemical synthesis. Simple triterpenes are components of All rights reserved surface waxes and specialized membranes and may potentially act as signal- ∗ Present address: Michael Smith Laboratories, ing molecules, whereas complex glycosylated triterpenes (saponins) provide University of British Columbia, Vancouver V6T 1Z4, Canada protection against pathogens and pests. Simple and conjugated triterpenes have a wide range of applications in the food, health, and industrial biotechnology sectors. Here, we review recent developments in the ﬁeld of triterpene biosynthesis, give an overview of the genes and enzymes that have been identiﬁed to date, and discuss strategies for discovering new triterpene biosynthetic pathways.

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Contents INTRODUCTION...... 226 TRITERPENEBIOSYNTHESIS...... 228 Cyclization of 2,3-Oxidosqualene: One Substrate, an Array of Products...... 228 Characterized Oxidosqualene Cyclases...... 230 Structural and Functional Analysis of Oxidosqualene Cyclases...... 233 Phylogenetic Analysis of Functionally Characterized Plant Oxidosqualene Cyclases ...... 235 OxygenationoftheTriterpeneScaffold...... 237 TriterpeneGlycosylation...... 239 Other Tailoring Enzymes ...... 241 STRATEGIES FOR DISCOVERING NEW OXIDOSQUALENE CYCLASESANDTRITERPENEPATHWAYS...... 244 Oxidosqualene Cyclase Discovery ...... 244 Oxidosqualene Cyclase Functional Analysis ...... 246 Identiﬁcation of Tailoring Enzymes ...... 246 CONCLUSIONS...... 248

INTRODUCTION Sterols and triterpenes are isoprenoids that are synthesized via the mevalonate pathway (21). The last common intermediate for their two pathways is 2,3-oxidosqualene. Sterols are important structural components of membranes and also have roles in signaling (as steroidal hormones). In contrast, triterpenes are not regarded as essential for normal growth and development, and although they do exist in plants in simple unmodiﬁed form, they often accumulate as conjugates with carbo- hydrates and other macromolecules, most notably as triterpene glycosides. Triterpene glycosides have important ecological and agronomic functions, contributing to pest and pathogen resistance and to food quality in crop plants. They also have a wide range of commercial applications in the food, cosmetics, pharmaceutical, and industrial biotechnology sectors (6, 88, 99, 119, 147). The cyclization of squalene (in bacteria) or 2,3-oxidosqualene (in fungi, animals, and plants) to sterol or triterpene products is one of the most complex enzymatic reactions known in terpene metabolism (1, 104, 153). These reactions have intrigued organic chemists and biochemists for

Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org the past half century (26). For the purposes of this review, we make a distinction between sterols and triterpenes based on the way in which these molecules are synthesized. In sterol biosynthesis,

Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. 2,3-oxidosqualene is cyclized to the sterols lanosterol (in fungi and animals) or cycloartenol (in plants) via the chair-boat-chair (CBC) conformation. In triterpene biosynthesis, in contrast, this substrate is folded into a different conformation—the chair-chair-chair conformation (CCC)— prior to cyclization into a huge array of triterpenes of diverse skeletal types, of which just one (β-amyrin) is shown as an example in Figure 1. The triterpenes are one of the largest classes of plant natural products, with more than 20,000 different triterpenes reported to date (47). The vast majority of triterpene diversity occurs in the plant kingdom, although other organisms also produce triterpenes. Examples include the synthesis of the simple triterpene hopene from squalene in bacteria (101) and the production of defense-related triterpene glycosides by sea cucumbers (146). Some other plant-derived specialized metabolites are synthesized via the CBC conformation, such as cucurbitacins (associated

226 Thimmappa et al. PP65CH08-Osbourn ARI 8 April 2014 21:43

Isopentenyl OPP OPP Dimethylallyl diphosphate + diphosphate (IPP) (DMAPP) FPS

OPP

Farnesyl pyrophosphate (FPP) SQS

SHC Hopanes (bacteria)

Squalene SQE Sterols Triterpenes

O O H 2,3-Oxidosqualene O CBC conformation CCC conformation

LAS CASCPQ BAS

H H

H H H H H HO HO HO HO H H H Lanosterol Cycloartenol Cucurbitadienol β-Amyrin

β -Sitosterol, Cucurbitacins Triterpene

Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org Ergosterol Cholesterol stigmasterol, glycosides brassinosteroids (e.g., glycyrrhizin, avenacins) Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. Fungi Animals Plants Plants Plants

Figure 1 The biosynthetic route to sterols and triterpenes. Sterols and triterpenes are synthesized via the mevalonic acid (MVA) pathway. The enzymes that catalyze the various steps are indicated in boxes. Enzyme abbreviations: FPS, farnesyl pyrophosphate synthase; SQS, squalene synthase; SQE, squalene monooxygenase or epoxidase; SHC, squalene-hopene cyclase; LAS, lanosterol synthase; CAS, cycloartenol synthase; CPQ, cucurbitadienol synthase; BAS, β-amyrin synthase. Other abbreviations: CBC, chair-boat-chair; CCC, chair-chair-chair.

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with the bitter taste of many members of the Cucurbitaceae family), which are synthesized from curcurbitadienol (130) (Figure 1), and tomato steroidal glycoalkaloids, which are synthesized via cholesterol (54). More than 100 different triterpene scaffolds are currently known in plants (163). Triterpene cyclization can thus lead to a wide array of different triterpene structures, all derived from the simple and ubiquitous linear isoprenoid substrate 2,3-oxidosqualene. These triterpene scaffolds can then provide the foundation for further modiﬁcation by triterpene-modifying (or tailoring) enzymes (e.g., cytochrome P450s, sugar transferases, and acyltransferases), thereby leading to enormous structural diversity. Nature’s triterpene reservoir remains largely undiscovered, despite the considerable commercial interest in these compounds for a range of applications (6, 88, 99, 119). Major advances in our understanding of the genes, enzymes, and pathways required to synthesize these molecules are now opening up unprecedented opportunities for triterpene metabolic engineering and for the discovery of new pathways and chemistries, facilitated by the recent discovery that the genes for triterpene pathways are—in at least some cases—organized in biosynthetic gene clusters in plant genomes (28, 29, 63, 69, 107).

TRITERPENE BIOSYNTHESIS

Cyclization of 2,3-Oxidosqualene: One Substrate, an Array of Products Cyclization of 2,3-oxidosqualene is catalyzed by enzymes known as oxidosqualene cyclases (OSCs), which generate either sterol or triterpene scaffolds in a process involving (a) substrate binding and preorganization (folding), (b) initiation of the reaction by protonation of the epoxide, (c) cyclization and rearrangement of carbocation species, and (d ) termination by deprotonation or water capture to yield a ﬁnal terpene product (Figure 2). Although variation in carbocation cyclization and rearrangement steps contributes substantially to scaffold diversity, the initial substrate folding step is critical, because this predisposes the substrate to follow a particular cyclization pathway. For example, the CBC conformation organizes cyclization to form the protosteryl cation, which then gives rise to sterols, whereas the CCC conformation directs cyclization into the dammarenyl cation, which then gives rise to a host of diverse triterpene scaffolds. In the synthesis of triterpenes in bacteria, squalene is cyclized to pentacyclic hopene by squalene-hopene cyclases (SHCs) (1). Following substrate folding, SHCs and OSCs initiate the cyclization reaction by protonation of −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→

Figure 2 Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org Oxidosqualene cyclization mechanisms. (a) The chair-boat-chair (CBC) conformation. All sterols derive from the protosteryl cation via the initial cyclization of the CBC conformation of 2,3-oxidosqualene. Following substrate folding, a protonation event opens the

Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. epoxide ring to generate a carbocation, which then triggers a series of electrophilic attacks by nearby double bonds in a cascade of C-C ring-forming reactions. Speciﬁcally, cyclization of 2,3-oxidosqualene to lanosterol proceeds through a series of carbocationic intermediates (C-2 → C-6 → C-10 → C-14 → C-20) to form the tetracyclic C-20 protosterol cationic intermediate (163). This intermediate then undergoes a further series of distinct hydride and methyl migrations that lead to the formation of either lanosterol (in fungi and animals) or cycloartenol (in plants). The protosteryl cation may also undergo alternative rearrangements to form molecules that are regarded as specialized metabolites, such as cucurbitadienol. Thus, in plants, the CBC conformation can also give rise to scaffolds other than cycloartenol that are associated with specialized metabolism but are not intermediates in primary sterol synthesis. (b) The chair-chair-chair (CCC) conformation. The majority of triterpene scaffold diversity derives from cyclization of the CCC conformation of 2,3-oxidosqualene, leading to the initial cyclization to the C-20 dammarenyl cation. Alternative rearrangements of this cation may then ensue, including ring expansion and methyl and hydride shifts that move the cation through a series of carbocationic intermediates (C-13 → C-14 → C-8 → C-9 → C-10 → C-5 → C-4), generating a variety of skeletal intermediates along the way (1–3, 163). The numbering of the carbon atoms in the pentacyclic triterpenes and the reference system for the rings (A–E) are shown at the bottom left.

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O 6 10 14 18 22 2 7 11 15 19 23 2,3-Oxidosqualene 3

+ a CBC Enz-AH 23 19 b CCC 14 6 Enz-AH+ 6 O 19 O 10 2 2 10 14 Prefolding and cyclization

1,2-hydride and H 20 H20 1,2-methyl shifts H

H Protosteryl H Dammarenyl HO HO H cation H cation Ring expansion H H 18 H H H Baccharenyl HO Baruol C-8 HO H8 H H cation HO HO cation H H Ring expansion Lanosterol 20 H H H HO Lupeol H H H Lupyl H H HO cation 9 C-9 H H HO H cation Ring

HO expansion 19 13 H Cucurbitadienol H Germanicyl H H H HO HO H cation H Ursanyl H α-Amyrin HO cation H H H

HO H Cycloartenol 13 H H Oleanyl H H HO cation HO H H β-Amyrin Skeletal rearrangement H H 14 H Taraxareyl H HO HO H cation H Taraxerol

H H 8 Multiflorenol H HO HO H cation H Multiflorenol Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org 9 H Walsurenyl cation HO H Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only.

H Companulyl 10 cation HO H

29 30 20 19 21 H HH H H 12 18 22 Glutinyl 11 13 17 5 25 26 HO Glutinol 28 HO cation 1 9 14 16 2 10 8 15 3 5 7 27 HO 4 6 H H 24 23 H H H Friedelyl HO H 4 cation O Friedelin

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the terminal double bond of squalene and the epoxide of 2,3-oxidosqualene, respectively. This protonation step deﬁnes SHC and OSC enzymes as class II terpene synthases. By contrast, class I terpene synthases (such as mono-, sesqui-, and some diterpene synthases) initiate cyclization through ionization of pyrophosphate with the assistance of Mg2+ cofactors (98).

Characterized Oxidosqualene Cyclases More than 80 OSCs have now been functionally characterized from plants, mostly by heterologous expression of cDNAs in appropriate yeast strains (Table 1). Approximately one-third of these are sterol synthases, a group that includes cycloartenol synthases as well as several lanosterol synthases. The function of lanosterol synthases in plants is unclear, but these enzymes appear to be involved to a small extent in the synthesis of phytosterols and potentially also steroid-derived metabolites (68, 97, 141). Some plant OSCs synthesize molecules other than cholesterol and lanosterol via the CBC fold; examples include cucurbitadienol synthase (CPX) from Cucurbita pepo (130) and parkeol synthase (OsPS) from rice (Oryza sativa) (56) (Table 1). The remaining OSCs synthesize triterpenes via the substrate CCC conformation (Figures 1 and 2). Collectively, plant OSCs are able to make a diverse array of triterpene scaffolds. Some make common scaffolds such as β- amyrin and lupeol, whereas others generate a host of other single or multiple 2,3-oxidosqualene cyclization products (Table 1). OSCs are encoded by multigene families in plants, so a single plant species is likely to be able to make multiple triterpene scaffolds. For example, the Arabidopsis thaliana genome contains 13 OSC genes, including genes for cycloartenol synthase (AtCAS1); lanosterol synthase (AtLSS1); a β-amyrin synthase (LUP4); several mixed-function OSCs that make β-amyrin, lupeol, and a range of other products; and other OSCs that produce “speciality” triterpenes such as marneral (MRN1), thalianol (THAS1), and arabidiol (PEN1) (22, 25, 27, 46, 52, 66–68, 72, 73, 77, 87, 126, 132, 135, 141, 161, 162) (Figure 3, Table 1). These 13 OSCs have different expression patterns and make different major products (Figure 3, Table 1), suggesting that they have specialized functions. BARS1 from A. thaliana makes baruol as its predominant cyclization product (∼90% abundance) but also makes 22 additional products (all at <2% abundance) (77) (Table 1). It is remarkable that BARS1 is able to make a total of 23 diverse cyclic products, including monocycles (6), bicycles (6/6), tricycles (6/6/5), tetracycles (6/6/6/5 and 6/6/6/6), and pentacycles (6/6/6/6/5 and 6/6/6/6/6). The BARS1 active site is thus able to deprotonate numerous sites from the A to the E ring, possibly at 14 distinct sites (77). The formation of multiple products by BARS1 may reﬂect the failure of this enzyme to precisely control cyclization toward baruol. The function of OSCs may be to prevent alternative cyclization paths rather than to stabilize particular intermediates

Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org that are being directed toward predetermined products—a concept known as negative catalysis (111). Thus, mechanistic diversity may be the default for triterpene cyclization, and product

Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. accumulation may result from the exclusion of alternative pathways (77). A friedelin synthase (FRS) has recently been cloned from Kalanchoe daigremontiana (150). FRS is able to span the maximum range of possible skeletal rearrangement from the dammarenyl cyclization pathway (Figure 2). Movement of the positive charge from the C-20 to the C-2 position involves the maximum possible number of 1,2 shifts (10 in total). When the cation reaches the C-2 position, it is attacked by the 3β-OH group to give a ketone group at C-3. Friedelin is therefore one of the most highly rearranged triterpenes known in plants, and unlike most other pentacyclic triterpenes, it lacks a double bond. Most triterpene synthases follow the standard route of C-C ring-forming and skeletal rearrangement reactions, in the process establishing several stereocenters and forming triterpene alcohols by introduction of a 3β-OH group and a double bond. However, FRS is not the only

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Table 1 Plant oxidosqualene cyclases (OSCs) GenBank ID/gene Ring OSC Species number Product Fold system Reference(s) LSS1 Arabidopsis thaliana At3g45130 Lanosterol CBC 6/6/6/5 68 LAS Lotus japonicus AB244671 Lanosterol CBC 6/6/6/5 118 PNZ1 Panax ginseng AB009031 Lanosterol CBC 6/6/6/5 141 CAS1 Abies magniﬁca AF216755 Cycloartenol CBC 6/6/6/5 — ACX Adiantum capillus-veneris AB368375 Cycloartenol CBC 6/6/6/5 137 CAS1 Arabidopsis thaliana At2g07050 Cycloartenola CBC 6/6/6/5 22 ∗, CS1 Avena strigosa AJ311790 Cycloartenol a CBC 6/6/6/5 40 BPX1 Betula platyphylla AB055509 Cycloartenol CBC 6/6/6/5 167 BPX2 Betula platyphylla AB055510 Cycloartenol CBC 6/6/6/5 167 ∗ CAS1 Chlamydomonas reinhardtii EDP09612 Cycloartenol CBC 6/6/6/5 80 CsOSC1 Costus speciosus AB058507 Cycloartenol CBC 6/6/6/5 61 CPX Cucurbita pepo AB116237 Cycloartenola CBC 6/6/6/5 130 CAS1 Dioscorea zingiberensis AM697885 Cycloartenol CBC 6/6/6/5 — CAS1 Glycyrrhiza glabra AB025968 Cycloartenola CBC 6/6/6/5 45 KdCAS Kalanchoe daigremontiana HM623872 Cycloartenol CBC 6/6/6/5 150 CAS Kandelia candel AB292609 Cycloartenol CBC 6/6/6/5 8 OSC5 Lotus japonicus AB181246 Cycloartenol CBC 6/6/6/5 120 CAS1 Luffa cylindrica AB033334 Cycloartenol CBC 6/6/6/5 42 OSC2 Oryza sativa AK121211 Cycloartenol CBC 6/6/6/5 56 PNX Panax ginseng AB009029 Cycloartenol CBC 6/6/6/5 71 CASPEA Pisum sativum D89619 Cycloartenol CBC 6/6/6/5 86 CAS Polypodiodes niponica AB530328 Cycloartenol CBC 6/6/6/5 — CAS Rhizophora stylosa AB292608 Cycloartenol CBC 6/6/6/5 8 RcCAS Ricinus communis DQ268870 Cycloartenola CBC 6/6/6/5 35 CPQ Cucurbita pepo AB116238 Cucurbitadienol CBC 6/6/6/5 130 OsPS Oryza sativa AK066327 Parkeol CBC 6/6/6/5 56 OEA Olea europaea AB291240 α-Amyrin CCC 6/6/6/6/6 112 LUP4 Arabidopsis thaliana At1g78950 β-Amyrin CCC 6/6/6/6/6 132 bAS Artemisia annua EU330197 β-Amyrin CCC 6/6/6/6/6 62 OXA1 Aster sedifolius AY836006 β-Amyrin CCC 6/6/6/6/6 14

Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org bAS1 Avena strigosa AJ311789 β-Amyrinb CCC 6/6/6/6/6 40 BPY Betula platyphylla AB055512 β-Amyrin CCC 6/6/6/6/6 167

Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. bAS Bruguiera gymnorrhiza AB289585 β-Amyrin CCC 6/6/6/6/6 8 AS Euphorbia tirucalli AB206469 β-Amyrin CCC 6/6/6/6/6 60 bAS1 Glycyrrhiza glabra AB037203 β-Amyrin CCC 6/6/6/6/6 44 AMY1 Lotus japonicus AB181244 β-Amyrin CCC 6/6/6/6/6 120 AMYI Medicago truncatula AJ430607 β-Amyrina CCC 6/6/6/6/6 57 bAS1 Nigella sativa FJ013228 β-Amyrina CCC 6/6/6/6/6 124 PNY1 Panax ginseng AB009030 β-Amyrin CCC 6/6/6/6/6 71 PNY2 Panax ginseng AB014057 β-Amyrin CCC 6/6/6/6/6 71 (Continued )

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Table 1 (Continued ) GenBank ID/gene Ring OSC Species number Product Fold system Reference(s) PSY Pisum sativum AB034802 β-Amyrin CCC 6/6/6/6/6 86 bAS Polygala tenuifolia EF107623 β-Amyrin CCC 6/6/6/6/6 — TTS1 Solanum lycopersicum HQ266579 β-Amyrinc CCC 6/6/6/6/6 149 BS Vaccaria hispanica DQ915167 β-Amyrin CCC 6/6/6/6/6 79 BPW Betula platyphylla AB055511 Lupeol CCC 6/6/6/6/5 167 LUS Bruguiera gymnorrhiza AB289586 Lupeol CCC 6/6/6/6/5 8 LUS1 Glycyrrhiza glabra AB116228 Lupeold CCC 6/6/6/6/5 45 KdLUS Kalanchoe daigremontiana HM623871 Lupeolc CCC 6/6/6/6/5 150 OSC3 Lotus japonicus AB181245 Lupeold CCC 6/6/6/6/5 120 OEW Olea europaea AB025343 Lupeol CCC 6/6/6/6/5 136 RcLUS Ricinus communis DQ268869 Lupeole CCC 6/6/6/6/5 35 TRW Taraxacum ofﬁcinale AB025345 Lupeol CCC 6/6/6/6/5 136 MRN1 Arabidopsis thaliana At5g42600 Marneralb CB —/6 162 THAS1 Arabidopsis thaliana At5g48010 Thalianolb CCC 6/6/5 27 SHS1 Aster tataricus AB609123 Shiononea CCC 6/6/6/6 121 KdGLS Kalanchoe daigremontiana HM623869 Glutinolc CCC 6/6/6/6/6 150 KdFRS Kalanchoe daigremontiana HM623870 Friedelinc CCC 6/6/6/6/6 150 KdTAS Kalanchoe daigremontiana HM623868 Taraxerolc CCC 6/6/6/6/6 150 IMS1 Luffa cylindrica AB058643 Isomultiﬂorenol CCC 6/6/6/6/6 43 OsIAS Oryza sativa AK067451 Isoarborinol CBC 6/6/6/6/5 164 PNA Panax ginseng AB265170 Dammarenediol II CCC 6/6/6/5 144 BOS Stevia rebaudiana AB455264 Baccharis oxide CCC 6/6/6/6 134 BARS1 Arabidopsis thaliana At4g15370 Mixed CCC 6/6/6/6 77 + products1 22other minor ones CAMS1 Arabidopsis thaliana At1g78955 Mixed products2,3,4 CCC 67 , , , , LUP1 Arabidopsis thaliana At1g78970 Mixed products5 4 6 8 9 CCC 6/6/6/6/5 46 LUP2 Arabidopsis thaliana At1g78960 Mixed CCC 73 products4,8,7,10,11,12,13,9,14 , LUP5 Arabidopsis thaliana At1g66960 Mixed products7 UC CCC 6/6/6/5 25 PEN1 Arabidopsis thaliana At4g15340 Mixed products15,16 CCC 6/6/5 66, 161

Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org PEN3 Arabidopsis thaliana At5g36150 Mixed CCC 87 , , , , , products17 12 18 19 20 21 11,10,14,UC

Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. PEN6 Arabidopsis thaliana At1g78500 Mixed products CCC 25 , , , CsOSC2 Costus speciosus AB058508 Mixed products10 6 4 UC CCC 61 , , MS Kandelia candel AB257507 Mixed products10 14 4 CCC 7 AMY2 Lotus japonicus AF478455 Mixed products10,4,UC CCC 57 , OSC8 Oryza sativa AK070534 Mixed products22 UC CCC 6/— 56 OSCPSM Pisum sativum AB034803 Mixed CCC 85 products14,4,23,9,12,10,6,8 (Continued )

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Table 1 (Continued ) GenBank ID/gene Ring OSC Species number Product Fold system Reference(s) RsM1 Rhizophora stylosa AB263203 Mixed products6,4,10 CCC 8 , , RsM2 Rhizophora stylosa AB263204 Mixed products24 4 10 CCC 8 TTS2 Solanum lycopersicum HQ266580 Mixed products14,4,23 CCC 6/6/6/6/6 149

Superscript lowercase letters denote respective OSC transcript abundance in specific tissues as determined by reverse transcription polymerase chain reaction (RT-PCR), semiquantitative PCR, or northern blot analysis: aall tissues; broot tip (epidermis); cleaf and fruit epidermis; dnodules; eepicuticular layers of leaves and stems. Superscript numbers denote the products of OSCs that generate mixed products, with the numbers for each OSC listed in the order of the products’ abundance in gas chromatography–mass spectrometry (most abundant product listed first): 1baruol; 2camelliol C; 3achilleol A; 4β-amyrin; 53β,20-dihydroxylupane; 6germanicol; 7tirucalla-7,21-dien-3β-ol; 8taraxasterol; 9-taraxasterol; 10lupeol; 11bauerenol; 12butyrospermol; 13multiflorenol; 14α-amyrin; 15(3S,13R)-malabarica-17,21-dien-3,14-diol (arabidiol); 16(3S,13R,21S)-malabarica-17-en-20,21-epoxy-3,14-diol (arabidiol 20,21-epoxide); 17tirucalla-7,24-dien-3-ol; 18tirucallol; 19isotirucallol; 2013H-malabarica-14(27),17,21-trien-3-ol; 21dammara-20,24-dien-3-ol; 22achilleol B; 23δ-amyrin; 24taraxerol; UCadditional uncharacterized products. Asterisks denote a predicted product. Abbreviations: CB, chair-boat; CBC, chair-boat-chair; CCC, chair-chair-chair.

exception to the rule. Some other plant triterpene synthases produce different types of scaffolds through unusual reactions such as formation of a ketone at the C-3 position (96, 121), introduction of an oxide bridge (134), or ring cleavage to give seco-triterpenes (“seco” is derived from “sec,” the Latin for “to cut”) (24, 135, 162) (Figure 4a–c). Triterpene synthases can also be made to generate different products following incubation with unnatural substrates. For example, the A. thaliana OSC LUP1 is a multifunctional triterpene synthase that normally converts 2,3-oxidosqualene to lupeol, pentacyclic triterpenes, and a variety of triterpene alcohols and diols. Incubation of LUP1 with the alternative substrate 2,3-22,23-dioxidosqualene, generated by squalene monooxygenase– mediated synthesis using 2,3-oxidosqualene as a substrate, results in the formation of other diverse heterocyclic structures that are produced via the epoxy dammarenyl cation (126, 129) (Figure 4d ).

Structural and Functional Analysis of Oxidosqualene Cyclases Crystal structures for plant sterol- or triterpene-synthesizing OSCs are not yet available. However, the structures of two other relevant enzymes—SHC from the bacterium Alicyclobacillus acidocal- darius (110, 154, 155) and human lanosterol synthase (LAS) (145)—have been experimentally determined. Although these two enzymes share only ∼25% amino acid identity, they have very

Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org similar architectures, both having two highly conserved (αα) barrel domains (known as the βγ fold) and a hydrophobic membrane-insertion helix (16, 98) (Figure 5). The substrate and cy-

Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. clization product likely enter and leave the active site of the enzyme via the membrane (110, 145). Investigations of SHC and LAS have led to the identiﬁcation of residues implicated in the initiation of cyclization, ring formation, and potential stabilization of carbocationic intermediates (1, 125, 156, 158–160). For example, alanine-scanning mutagenesis of the aromatic residues in the hydrophobic cavity of SHC resulted in altered product proﬁles, thereby establishing the roles of different residues in product formation (84, 117). Site-saturation mutagenesis of the termination residue His234 in yeast ergosterol synthase (His232 in human LAS) also resulted in a range of cyclic products (159). This residue has been postulated to deprotonate the tetracyclic C-8/9 cation in lanosterol and cycloartenol synthases. Plant triterpene synthases such as lupeol and β-amyrin synthase have an aromatic residue at the corresponding position, which has been proposed to

www.annualreviews.org • Triterpene Biosynthesis in Plants 233 PP65CH08-Osbourn ARI 8 April 2014 21:43 OSC H -ol β H H H Baruol H BARS1 PEN6 H A. thaliana CAMS1 Bauerenol Camelliol C LUP5 ) The major b H HO HO HO H H Tirucalla-7,21-dien-3 ). HO H H LUP4 H -Amyrin Table 1 H β H MRN1 H HO PEN3 H H HO Tirucalla-7,24-dien-3-ol HO H O LUP1 H Lupeol H H H HO ) Heat map showing expression proﬁles for 12 of the 13 a H CAS1 LSS1 H Cycloartenol Lanosterol HO H LUP2 PEN1 H -Amyrin THAS1 Arabidiol Thalianol Marneral β HO OSCs. Some of these OSCs also make other products ( HO Gene expression (%) 100 0 HO HO

A. thaliana

PEN3 PEN3 oxidosqualene cyclases (OSCs). (

PEN1 PEN1

BARS1 BARS1

MRN1 MRN1

THAS1 THAS1

PEN6 PEN6

LUP4 LUP4

Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org LUP1 Arabidopsis thaliana

LUP2 LUP2 LUP5 LUP5

Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. LSS1 LSS1 CAS1 CAS1 were not detected, and so this gene was not included). Expression data were retrieved from Genevestigator V3 (48). ( CAMS1 Seedling Inflorescence Raceme Flower Stamen Anther Pollen Abscission zone Pistil Carpel Stigma Ovary Petal Sepal Pedicel Silique Seed Embryo Endosperm Testa Pericarp Shoot Roots Primary root Root tip Elongation zone Maturation zone Stele Pericycle Lateral root Roots Shoot Seedling and seeds ab Inflorescence Figure 3 Expression proﬁles and major products of 2,3-oxidosqualene cyclization products made by each of the 13 genes (transcripts for

234 Thimmappa et al. PP65CH08-Osbourn ARI 8 April 2014 21:43

stabilize the intermediary cation, thereby enabling further cyclization and ring expansion to pentacyclic triterpenes (109). In other studies, Matsuda and coworkers (76) combined two mutations (His477Asn and Ile481Val) to successfully convert the A. thaliana cycloartenol synthase CAS1 into an accurate lanosterol synthase, and Ebizuka and coworkers (72) used a combination of domain swapping and site-directed mutagenesis with lupeol and β-amyrin synthases to identify a motif (MWCYCR) within an 80-amino-acid region that determines β-amyrin formation. Two of these residues (Trp259 and Tyr261) are implicated in D/E-ring stabilization. These examples are not exhaustive, but they serve to illustrate the potential of combining sequence alignments and homol- ogy modeling with mutagenesis to probe the mechanisms of this fascinating family of enzymes. Investigations of the ways in which these enzymes generate such metabolic diversity from a single linear substrate will be a fertile area for future investigation, for both fundamental science and biotechnological applications. This would be greatly facilitated by experimental determination of the structures of exemplar plant sterol and triterpene synthases.

Phylogenetic Analysis of Functionally Characterized Plant Oxidosqualene Cyclases Figure 6 shows a phylogenetic tree illustrating the relatedness of characterized plant OSCs. It is ev- ident that the OSCs that generate the protosterol cation—the precursor for the sterols cycloartenol and lanosterol—all group together. These include OSCs from both dicots and monocots. The triterpene synthases that generate the C-20 dammarenyl cation all group separately from the sterol synthases. These enzymes most likely arose directly or indirectly by duplication and divergence of cycloartenol synthase genes (28, 29, 53, 104, 107, 109, 164). It follows that the triterpene CCC fold and associated ability to cyclize 2,3-oxidosqualene to the dammarenyl cation are likely to be derived from the CBC fold of a sterol-producing progenitor, and that changes in substrate folding underlie this major functional diversiﬁcation event. Four of the characterized triterpene synthases are from monocots: OsIAS, isoarborinol synthase from rice (Oryza sativa) (164); OsOSC8, also from rice, which makes achilleol B as its main cyclization product along with a variety of other triterpenes (56); β-amyrin synthase from diploid oat (Avena strigosa), which is required for the synthesis of the antimicrobial β-amyrin-derived triterpene glycosides known as avenacins (40, 107); and the multifunctional enzyme CsOSC2 from crape ginger (Costus speciosus), which generates β-amyrin, lupeol, germanicol, and additional uncharacterized cyclization products (61). These monocot triterpene synthases are more similar to the sterol synthases when compared with the other triterpene synthases shown in Figure 6, which are all from dicots, and form a discrete subgroup. It was previously reported that oat β-amyrin synthase (the only charac-

Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org terized β-amyrin synthase from monocots) evolved independently of the dicot β-amyrin synthases and shares greater similarity with the cycloartenol synthases (40, 107). This monocot β-amyrin

Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. synthase is clearly distinct from the dicot β-amyrin synthases, which form a large, distinct clade elsewhere in the tree (Figure 6). Thus, the ability to cyclize 2,3-oxidosqualene to β-amyrin has arisen more than once in the plant kingdom. One other monocot OSC groups with the monocot triterpene synthase clade, namely O. sativa parkeol synthase (OsPS), which, like cucurbitadienol synthase from C. pepo, generates a sterol cyclization product (in this case parkeol) (56). This contrasts with the dicot OSC cucurbitadienol synthase from C. pepo (CpCPQ), which makes cucurbitadienol, an alternative rearrangement product of the protosteryl cation (130). CPQ groups with the cycloartenol and lanosterol synthases (Figure 6). The remainder of the OSCs shown in Figure 6 consist of a discrete clade of lupeol synthases; a clade of A. thaliana OSCs that make particular major products, in some cases along with multiple minor products (AtBARS1, AtPEN1, AtTHAS1, AtPEN3, ArPEN6, and AtMRN1) (25, 27, 77,

www.annualreviews.org • Triterpene Biosynthesis in Plants 235 PP65CH08-Osbourn ARI 8 April 2014 21:43

a Introduction of ketone functionality SHS

H 18 H H H H H H

H 3 O 3 HO 4 HO 4 H HO Baccharenyl cation C-4 cationShionone Shionone (enol form) b Introduction of an oxide bridge BOS

H 18 H

H 10 H O H-O H H Baccharenyl cation Baccharis oxide

c Ring breaking MRN1

O H 8 5 H-O H Bicylic C-8 tertiary cation Marneral

PEN6

13 13 H H H 14 14 H 8 H 8 H HO HO HO H H H H Oleanyl cation β-Seco-amyrin

d Generation of heterocyclic triterpenes HO LUP1 O O O H H

Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org H SQE LUP1 H

H H H O HO HO HO Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. O H H H 2,3-Oxidosqualene 2,3-22,23-Dioxidosqualene Epoxydammarenyl Epoxy baccharene Oxacyclic cation cation triterpene diol

O H

H H HO H Dammarenediol

236 Thimmappa et al. PP65CH08-Osbourn ARI 8 April 2014 21:43

γ γ

β β

Human LAS Bacterial SHC Figure 5 Crystal structures of human lanosterol synthase (LAS) (Protein Data Bank ID 1w6K) and bacterial squalene-hopene cyclase (SHC) (Protein Data Bank ID 2sqc). These enzymes have the βγ fold typical of class II terpene synthases (β and γ domains shown in green and yellow, respectively) and a hydrophobic membrane-insertion helix (shown in red ) that is conserved across LAS, SHC, and other oxidosqualene cyclases.

87, 161, 162); and a broad group of OSCs, many of them multifunctional, that make a range of cyclization products (see Table 1 for references). Multifunctional OSCs may conceivably represent evolutionary intermediates (104) that are in the process of being reﬁned to become more accurate pentacyclic triterpene synthases. As more characterized OSCs emerge, we will be able to draw on and augment this phylogenetic framework and thereby continue to develop our ability to predict product proﬁles based on DNA sequences.

Oxygenation of the Triterpene Scaffold Although simple triterpenes such as β-amyrin and lupeol are common in plants, triterpene scaffolds are often further modiﬁed to more elaborate molecules by tailoring enzymes (6, 88, 99, 119). Cytochrome P450–mediated oxygenation of the scaffold (e.g., introduction of hydroxyl, ketone, aldehyde, carboxyl, or epoxy groups) is common. The functional groups introduced by such modiﬁcations may then pave the way for further tailoring by enzymes such as sugar transferases and acyltransferases. P450s therefore play a key role in functionalizing the triterpene scaffold.

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

Figure 4

Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org Synthesis of unusual triterpene skeletons. (a) Introduction of ketone functionality. Shionone synthase (SHS) makes shionone, a major triterpene found in roots of members of the Asteraceae family (96, 121). Shionine is a triterpene ketone generated via a tetracyclic baccharenyl secondary cation. (b) Introduction of an oxide bridge. Baccharis oxide synthase (BOS), an oxidosqualene cyclase (OSC) Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. from Stevia rebaudiana, forms baccharis oxide through a series of 1,2-hydride and 1,2-methyl shifts in the baccharenyl cation that lead to formation of a C-10 cation. Intramolecular attack of the 3β-hydroxyl onto the C-10 cation then results in a 3,10-oxide with a simultaneous chair-to-boat conformational change in the AB rings (134). Thus, BOS is able to introduce an oxide bridge, a modiﬁcation that normally requires the action of a cytochrome P450 oxidoreductase. (c) Ring breaking, showing OSCs that make seco-triterpenes. Arabidopsis thaliana marneral synthase (MRN1) makes the iridal-type triterpene marneral, which has an A-ring seco structure. Cyclization of 2,3-oxidosqualene leads to a bicyclic (6/6) C-8 tertiary cation in a chair-boat conformation. Following the 1,2 shifts, C-8 moves to C-5. A ring cleavage results from attack of the 3β-OH group on the cation, leading to 3,4-seco-aldehyde (162). In another ring-breaking reaction, A. thaliana seco-β-amyrin synthase (PEN6) makes C-ring seco-α-amyrin along with α-andβ-amyrin (24, 135). Seco-β-amyrin is made via a C-13 oleanyl cationic intermediate (24, 135). (d ) Generation of heterocyclic triterpenes. The A. thaliana OSC LUP1 is a multifunctional triterpene synthase that converts 2,3-oxidosqualene to lupeol, pentacyclic triterpenes, and a variety of triterpene alcohols and diols. Incubation of LUP1 with the alternative 2,3-22,23-dioxidosqualene leads to formation of other diverse heterocyclic structures generated via the epoxy dammarenyl cation (126, 129).

www.annualreviews.org • Triterpene Biosynthesis in Plants 237 PP65CH08-Osbourn ARI 8 April 2014 21:43

Lupeol synthases Rice parkeol *AtBARS1-At4g15370 synthase (PS) AtPEN1-At4g15340 AtTHAS1-At5g48010*

PgPNA-AB265170 Monocot Diverse *AtPEN3-At5g36150 OeOEA-AB291240 triterpene synthases products AtMRN1-At5g42600 *AtPEN6-At1g78500 Oat β-amyrin synthase (AsbAS) * * RsM2-AB263204 * Lanosterol

83 synthases *KcMS-AB257507 93 BgLUS-AB289586 33 AsbAS1-AJ311789 BpOSCBPW-AB055511 OsIAS-AK067451 ToTRW-AB025345 LjOSC3-AB181245 100 OeOEW-AB025343 GgLUS1-AB116228 100 OsOSC8-AK07053 Cucurbitadienol RcLUS-DQ268869 OsPS-AK066327 LcIMS1-AB058643 31 synthase (CPQ) CsOSC2-AB058508

AtLUP2-At1g78960 7 100

99 * 8

AtLUP5-At1g66960 100 * 100 99

100 99 PgPNZ1-AB009031 100 AtLAS1-At3g45130 AtLUP1-At1g78970 100 LjOSC7AB244671 * 92

45 97 47 48 CpCPQ-AB116238PnCAS-AB530328 AtCAMS1-At1g78955 53 72 * 99 100 99 AcACX-AB368375 42 100 AmCAS1-AF216755 AtBAS-At1g78950 98 99 48 OsOSC2-AK121211 NsbAS1-FJ013228 30 100 AsCS1-AJ311790 82 35 50 89 CsOSC1-AB058507 KdGLS-HM623869 DzCAS1-AM697885 96 63 72 99 100 LcCAS1-AB033334 KdFRS-HM623870 100 99 Friedelin 35 CpCPX-AB116237 78 BpCASBPX1-AB055509 synthase KdLUS-HM623871 70 92 41 8721 100 KdCAS-HM623872 KdTAS-HM623868 100 RsCAS-AB292608 88 71 KcCAS-AB292609 31 29 RsM1-AB263203 34 AtCAS1-At2g07050 BpCASBPX2-AB055510 * 49 31 BgBAS-AB289585 22 RcCAS-DQ268870 Cycloartenol 100 99 PgOSCPNX1-AB009029 synthases EtAS-AB206469 48 80 PsCASPEA-D89619 GgCAS1-AB025968 59 LjOSC5-AB181246 BpOSCBPY-AB055512 7850 12 95 75 100 DdCAS1-DDB G0269226 PsOSCPSY-AB034802 Human LAS-AAC50184CrCAS1-EDP09612 46 2 MtbAS1-AJ430607 100

53 32 SaCAS-CAD39196SaLAS-EAU65610

LjOSC1-AB181244 99

100 GgbAS1-AB037203 0.1 PtbAS-EF107623

LjAMY2-AF478455 * VhBS-DQ915167 McLAS-AAU91075 * PsOSCPSM-AB034803SlTTS2-HQ266580SlTTS1-HQ266579

PgOSCPNY2-AB014057 AaBAS-EU330197 β PgOSCPNY1-AB009030

-Amyrin AsOXA1-AY836006

synthases StrBOS-AB455264 AtaSHS-AB609123 Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org

Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. Baccharis oxide Shionone synthase (BOS) synthase (SHS)

Figure 6 Neighbor-joining tree of oxidosqualene cyclases (OSCs) from diverse plant species. OSC amino acid sequences (Table 1) were aligned using ClustalW with default parameters as implemented in the program MEGA5 (143). All positions containing gaps and missing data were eliminated. Evolutionary distances were computed using the JTT matrix-based method (59). Evolutionary analyses were conducted in MEGA5 with 1,000 bootstrap replicates (143). The scale bar (bottom, center) indicates 0.1 amino acid substitutions per site. The outgroup sequences used were cycloartenol synthases from Chlamydomonas reinhardtii (GenBank ID EDP09612) (80), Dictyostelium discoideum (dictyBase ID DDB_G0269226) (34), and the methanotropic bacterium Stigmatella aurantiaca (GenBank ID CAD39196) (9) and lanosterol synthases from Homo sapiens (GenBank ID AAC50184) (145), Stigmatella aurantiaca (GenBank ID EAU65610) (9), and the methanotropic bacterium Methylococcus capsulatus (GenBank ID AAU91075) (75). Cycloartenol synthases are shown in blue; monocot and dicot β-amyrin synthases are shown in red. Multifunctional OSCs are marked with an asterisk.

238 Thimmappa et al. PP65CH08-Osbourn ARI 8 April 2014 21:43

The plant P450s are a large and diverse group of enzymes that have been assigned to 11 clades based on sequence similarity. The P450s within these clades are further subdivided into families (95). Until recently, very little was known about the types of P450 enzymes involved in triterpene modifications. Within the past five years, however, there has been considerable activity in this area, and a total of 20 triterpene-modifying P450s have now been reported, 19 of them from dicots (18, 20, 28, 29–32, 37, 38, 49, 69, 70, 108, 127, 128, 131) (Table 2). Most of those characterized so far modify β-amyrin-based scaffolds (Figure 7a), although P450s that oxygenate other triterpene scaffolds—such as α-amyrin, lupeol, dihydro-lupeol, dammarenediol II, thalianol, arabidiol, marneral, and marnerol—have also been reported (Figure 7b). The enzymes shown in Table 2 belong to the CYP51, CYP71, CYP72, and CYP85 clans. The expansion of the latter three clans in plants is associated with metabolic diversification as plants colonized land (36, 82, 95). In contrast, members of the CYP51 clan are normally highly conserved and are associated with primary sterol biosynthesis. The limited number of P450s characterized to date makes it difficult to draw conclusions about the origins of triterpene-modifying P450s, but recruitment of P450s for triterpene biosynthesis is likely to have occurred multiple times during evolution. The single example of a triterpene-modifying P450 from monocots [CYP51H10 from diploid oat (Avena strigosa)] belongs to the CYP51 clan (32, 108). The CYP51 clan is regarded as one of the most ancient of the P450 clans (95). Until recently, CYP51 enzymes were only known to have a single and highly conserved function—as sterol 14α-demethylases in the synthesis of essential sterols. The oat CYP51H10 enzyme is the first member of the CYP51 clan to have a different function—in the modification of the β-amyrin scaffold during the synthesis of antimicrobial triterpene glycosides known as avenacins. CYP51H10 belongs to a newly defined divergent group of CYP51 enzymes known as the CYP51H subfamily, which appears to be restricted to monocots and which also includes nine members of unknown function in rice (53, 108). The oat enzyme likely arose by duplication and neofunctionalization of a sterol 14α-demethylase gene (108). This enzyme is able to carry out two modifications to the β-amyrin scaffold (32, 70), specifically addition of a 16-hydroxyl group to the D ring and introduction of a 12-13 epoxide to the C ring (32). Molecular modeling and docking experiments suggest that C-16 hydroxylation precedes C-12,C- 13 epoxidation (32). The 12-13 epoxide group is critical for the antimicrobial activity of avenacins (32). Multifunctional P450 enzymes from other P450 families that catalyze both hydroxylation and epoxidation reactions were previously known in microbes. To our knowledge, however, the oat CYP51H10 enzyme is the first plant P450 to catalyze both of these modifications. Given the close biogenic relationship between sterols and triterpenes, it may well be that other members of the divergent CYP51H subfamily (such as those in rice) are able to modify triterpene scaffolds. Functional analysis of this P450 subfamily in monocots is likely to be a fertile area for future

Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org investigation.

Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. Triterpene Glycosylation Triterpenes are often present in plants in glycosylated form. Glycosylation results in increased polarity and is often associated with bioactivity (6). Glycosylated triterpenes are also referred to as saponins. Many triterpenes have one or more sugar chains, normally attached at the C-3 and/or C-28 positions (although glycosylation at the C-4, C-16, C-20, C-21, C-22, and/or C-23 positions can also occur). These sugar chains are usually composed of glucose, galactose, arabinose, rhamnose, xylose, and glucuronic acid (although other sugars may also be incorporated) and are added onto hydroxyl groups or carboxyl groups, forming sugar acetals and sugar esters, respectively (147). Oligosaccharide chain formation is believed to occur through successive addition of sugars rather than transfer of a preformed sugar chain en bloc (6, 99, 119), and the

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Table 2 Triterpene-modifying cytochrome P450s GenBank Clan Gene Species ID Substrate Reaction Reference(s) CYP51 CYP51H10 Avena strigosa ABG88961 β-Amyrin C-16 hydroxylation, 32, 70, 108 C-12–C-13 epoxidation CYP71 CYP71A16 Arabidopsis thaliana NP_199073 Marneral, marnerol C-23 hydroxylation 20, 28 CYP71D353 Lotus japonicus KF460438 Dihydrolupeol, C-20 hydroxylation, 69 20-hydroxylupeol C-28 oxidation (three-step oxidation) CYP93E1 Glycine max BAE94181 β-Amyrin, C-24 hydroxylation 131 sophoradiol CYP93E2 Medicago truncatula ABC59085 β-Amyrin C-24 hydroxylation 30 CYP93E3 Glycyrrhiza BAG68930 β-Amyrin C-24 hydroxylation 127 uralensis CYP705A1 Arabidopsis thaliana NP_193268 Arabidiol C-15–C-16 cleavage 20 CYP705A5 Arabidopsis thaliana Q9FI39 7β-Hydroxythalianol C-15–C-16 29 desaturation CYP72 CYP72A61v2 Medicago truncatula BAL45199 24-Hydroxy-β- C-22 hydroxylation 31, 128 amyrin CYP72A63 Medicago truncatula BAL45200 β-Amyrin C-30 hydroxylation, 128 C-30 oxidation (three-step oxidation) CYP72A68v2 Medicago truncatula BAL45204 Oleanolic acid C-23 hydroxylation 31, 128 (three-step oxidation) CYP72A154 Glycyrrhiza BAL45207 β-Amyrin, C-30 hydroxylation 128 uralensis 11-oxo-β-amyrin (β-amyrin); C-30, C-22, and C-29 oxidation (11-oxo-β-amyrin) CYP85 CYP88D6 Glycyrrhiza BAG68929 β-Amyrin, C-11 oxidation 127 uralensis 30-hydroxy-amyrin (two-step oxidation) CYP708A2 Arabidopsis thaliana Q8L7D5 Thalianol C-7 hydroxylation 20, 29 CYP716A12 Medicago truncatula ABC59076 β-Amyrin, C-28 oxidation 18, 30 α-amyrin, lupeol (three-step oxidation) CYP716A15 Vitis vinifera BAJ84106 β-Amyrin, C-28 oxidation 30 Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org α-amyrin, lupeol (three-step oxidation) CYP716A17 Vitis vinifera BAJ84107 β-Amyrin C-28 oxidation 30 Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. (three-step oxidation) CYP716A47 Panax ginseng AEY75217 Dammarenediol II C-12 hydroxylation 38 CYP716A53v2 Panax ginseng AFO63031 Protopanaxadiol C-6 hydroxylation 37 CYP716AL1 Catharanthus roseus AEX07773 β-Amyrin, C-28 oxidation 49 α-amyrin, lupeol (three-step oxidation)

240 Thimmappa et al. PP65CH08-Osbourn ARI 8 April 2014 21:43

recent demonstration of sequential glycosylation of steroidal alkaloids is consistent with this (54). The number of sugar chains, their composition, and their position on the triterpene scaffold provide considerable potential for metabolic diversification. The enzymes that have so far been shown to glycosylate triterpenes belong to Carbohydrate- Active Enzymes (CAZy) family 1, as defined by Cantarel et al. (15). The family 1 glycosyltransferases are one of the largest groups of natural product–decorating enzymes in higher plants. The expansion of this family in higher plants reflects chemical diversification during the adaptation of plants to life on land (17, 165). These glycosyltransferases transfer sugars from nucleotide diphosphate-activated sugar moieties, usually UDP-glucose, to small hydrophobic acceptor molecules, and are referred to as family 1 UDP glycosyltransferases (UGTs) (10, 148). Other sugar donors include UDP-galactose, UDP-rhamnose, UDP-xylose, UDP-glucuronate, and UDP- arabinose. UGTs can be either highly selective or promiscuous in terms of the range of acceptors they recognize. The major principle governing acceptor recognition by UGTs seems to be regiose- lectivity (systematic glycosylation of the same position) rather than specificity for one or several structurally related compounds (19, 39, 148). To date, 12 UGTs that are able to glycosylate triterpenes have been reported from various plant species (4, 5, 79, 94, 123, 131) (Table 3). Of these, 9 belong to the UGT73 family; the other 3 belong to the UGT71, UGT74, and UGT91 families, respectively. Figure 8 shows examples of the types of reactions catalyzed by these enzymes. These reactions include addition of glucose to the C-3 hydroxyl or the C-28 carboxylic acid groups of hederagenin and other oleanane scaffolds, as well as sugar-sugar additions such as transfer of a glucose onto a C-28 arabinose and additions of galactose and rhamnose to a C-3 sugar (4, 5, 123, 133) (Figure 8, Table 3). The majority of these enzymes are from the UGT73 family. It is noteworthy that other UGT73 enzymes use steroidal compounds as acceptors, including steroids and steroidal alkaloids (SaGT4A, SGT1, SGT2, and SGT3) and brassinosteroids (UGT73C5 and UGT73C6) (51, 54, 55, 64, 65, 83, 106, 139, 151, 152). The identification of further enzymes that are able to glycosylate triterpene scaffolds in different positions and build oligosaccharide chains will be important in manipulating the physicochemical and biological properties of these molecules.

Other Tailoring Enzymes Triterpene scaffolds can undergo various other modiﬁcations in addition to oxygenation and glycosylation. For example, antimicrobial triterpene glycosides that are produced in oat roots (avenacins) are acylated at the C-21 position with either N-methyl anthranilate or benzoate (see Figure 7a for the structure of the major oat root triterpene, avenacin A-1). Mugford et al. (92)

Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org recently showed that the serine carboxypeptidase-like acyltransferase AsSCPL1 is responsible for this acylation step. In contrast to the BAHD acyltransferases, which utilize coenzyme A (CoA)–

Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. thioesters as the acyl donor, SCPL-like acyltransferases use O-glucose esters (81, 90, 91, 138). The sugar donors used by AsSCPL1 are N-methyl anthranilate-O-glucose and benzoyl-O-glucose (92). N-Methyl anthranilate is synthesized from anthranilate by the anthranilate N-methyltransferase AsMT1 (89). The family 1 glycosyltransferase (AsUGT74H5) then catalyzes the glucosylation of N-methyl anthranilate to N-methyl anthranilate-O-glucose, and the related enzyme AsUGT74H6 converts benzoate to benzoyl-O-glucose (102). Intriguingly, the three genes encoding AsSCPL1, AsMT1, and AsUGT74H5 are immediately adjacent to one another and form part of a larger biosynthetic cluster for avenacin synthesis that also includes genes for the β-amyrin synthase AsBAS1 and the β-amyrin-modifying P450 AsCYP51H10 (32, 89, 92, 93, 102, 107, 108). C-21 acylation is a common feature of many of the most cytotoxic triterpene glycosides (105). Consistent with this, acylation is important for the potent biological activity of avenacins (89). This set of

www.annualreviews.org • Triterpene Biosynthesis in Plants 241 PP65CH08-Osbourn ARI 8 April 2014 21:43

CYP72A61v2 OH

HO HO OH OH 24-Hydroxy-β-amyrin Soyasapogenol B

CYP93E1 CYP93E2 CYP93E1 OH CYP93E3 CYP93E2 CYP93E3 HO Sophoradiol OH COOH

CYP72A63 CYP72A63 CYP72A154 HO HO 30-Hydroxy-β-amyrin 11-Deoxoglycyrrhetinic acid

COOH CYP88D6 O O CYP72A154 HO

β-Amyrin HO HO 11-Oxo-β-amyrin Glycyrrhetinic acid CYP51H10 O

O O

O O HNCH3 OH OH HO Glc Ara O CYP716A12 12,13β-Epoxy- Glc CYP716A15 β β OH CYP716A17 16 -hydroxyl- -amyrin Avenacin A-1 CYP716AL1

COOH CYP72A68v2 COOH

Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org HO HO HOOC Oleanolic acid Gypsogenic acid Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only.

Figure 7 Modiﬁcation of triterpene scaffolds by cytochrome P450s. (a) Characterized P450 enzymes that modify the β-amyrin scaffold. (b) Characterized P450 enzymes that modify other scaffolds. Multiple arrows indicate multiple sequential reactions catalyzed by the corresponding P450. The colors indicate the P450 clan to which each enzyme belongs: purple, CYP51; blue, CYP71; green, CYP72; red, CYP85. Table 2 provides more information about these enzymes.

242 Thimmappa et al. PP65CH08-Osbourn ARI 8 April 2014 21:43

b CYP716A12 CYP716A15 CYP716AL1 COOH

HO HO

α-Amyrin Ursolic acid

CYP716A12 CYP716A15 CYP716AL1 COOH

HO HO Lupeol Betulinic acid

HO HO

CYP71D353 CYP71D353 COOH

HO HO HO

Dihydro-lupeol 20-Hydroxy-lupeol 20-Hydroxy betulinic acid

OH OH OH OH OH

CYP716A47 CYP716A53v2 HO HO HO OH Dammarenediol II Protapanaxadiol Protapanaxatriol

HO CYP708A2 HO OH CYP705A5 HO OH Thalianol 7β-Hydroxythalianol Desaturated 7β-hydroxythalianol

HO O

CYP705A1 + HO HO OH X Arabidiol 14-Apo-arabidiol Unidentified side chain Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. O O CYP71A16 HO Marneral 23-Hydroxymarneral

CYP51

HO HO CYP71 CYP71A16 HO CYP72 CYP85 Marnerol 23-Hydroxymarnerol

Figure 7 (Continued )

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Table 3 Triterpene glycosyltransferases (GTs) GenBank Family GT Species ID Substrate Reaction Reference UGT71 UGT71G1 Medicago AAW56092 Hederagenin β-D-Glucosylation 4 truncatula UGT73 UGT73C10 Barbarea vulgaris AFN26666 β-Amyrin, C-3 5 hederagenin β-D-glucosylation UGT73C11 Barbarea vulgaris AFN26667 β-Amyrin, C-3 5 hederagenin β-D-glucosylation UGT73C12 Barbarea vulgaris AFN26668 β-Amyrin, C-3 5 hederagenin β-D-glucosylation UGT73C13 Barbarea vulgaris AFN26669 β-Amyrin, C-3 5 hederagenin β-D-glucosylation UGT73K1 Medicago AAW56091 Hederagenin, β-D-Glucosylation 4 truncatula soyasapogenol B/E UGT73F3 Medicago ACT34898 Hederagenin C-28 94 truncatula β-D-glucosylation UGT73F2 Glycine max BAM29362 Saponin A0-αg C-3 123 β-D-glucosylation UGT73F4 Glycine max BAM29363 Saponin A0-αg C-3 β-D-xylosylation 123 UGT73P2 Glycine max BAI99584 Soyasapogenol B C-2 133 3-O-glucuronide β-D-galactosylation UGT74 UGT74M1 Saponaria vaccaria ABK76266 Gypsogenic acid C-28 79 β-D-glucosylation UGT91 UGT91H4 Glycine max BAI99585 Soyasaponin III C-2 133 β-D-rhamnosylation

enzymes for the synthesis of the acyl donor and subsequent transfer of the acyl group onto the triterpene scaffold is therefore likely to be a useful resource for triterpene modiﬁcation.

STRATEGIES FOR DISCOVERING NEW OXIDOSQUALENE CYCLASES AND TRITERPENE PATHWAYS

Oxidosqualene Cyclase Discovery Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org Triterpenes are usually synthesized in particular plant tissues and/or at certain developmental

Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. stages. Production may also be induced in response to treatment with abiotic/biotic stresses or elicitors such as methyl jasmonate. For example, the triterpene glycoside saponins glycyrrhizin and avenacins accumulate in the roots of licorice (Glycyrrhiza)andoat(Avena), respectively, whereas in alfalfa (Medicago sativa), triterpene glycoside synthesis is induced in the leaves in response to pathogen and herbivore attack (6, 88, 99, 119). Treatment of cell suspension cultures with methyl jasmonate can provide a simple system for inducing and investigating triterpene biosynthesis in response to elicitor treatment (140). Most of the OSCs characterized to date have been cloned via expression-based strategies using degenerate primers and rapid ampliﬁcation of cDNA ends (RACE)–polymerase chain reaction (PCR) or by screening cDNA libraries (6). Recent developments in high-throughput transcriptomics methods now open up powerful strategies for gene discovery that can be applied to diverse plant species.

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COOH

Glc O UGT73C10 OH O COOH Hederagenin 3- -glucoside

OGlc OH Hederagenin UGT73F3 O HO

OH Hederagenin 28-O-glucoside

O Glc COOH UGT74M1 O

HO HO

COOH COOH Gypsogenic acid Gypsogenic acid 28-O-glucoside

OH OH

O Ara O Ara Glc UGT73F2 AGlc O AGlc O Gal Gal OH Glc OH Glc Soybean saponin A0-αg Soybean saponin A0-αg 3'-O-glucoside

OH OH OH UGT73P2 UGT91H4

AGlc O AGlc O AGlc O Gal Gal OH OH Rha OH Soyasapogenol B 3-O-glucuronide Soyasaponin III Soyasaponin I

Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org Figure 8 Reactions catalyzed by characterized triterpene glycosyltransferases. UGT73C10 from Barbarea vulgaris and UGT73F3 from Medicago

Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. truncatula glucosylate the C-3 and C-28 positions of β-amyrin-derived (oleanane) triterpenes, respectively (5, 94). The Saponaria vaccaria enzyme UGT74M1 glucosylates the C-28 position of another oleanane triterpene, gypsogenic acid (79). Three glycosyltransferases that add sugars to triterpene glycosides are also shown. UGT73F2 from soybean (Glycine max) glucosylates the C-22-linked arabinose of soybean saponin AO-αg (123); UGT73P2, also from soybean, adds a galactose to the C-3-linked glucuronic acid of soyasapogenol-B, and a second soybean enzyme, UGT91H4, then adds a rhamnose to the galactose moiety (133).

The completion of the ﬁrst plant genome sequence provided surprising insights into the potential of plants to make triterpenes. The A. thaliana genome contains 13 OSC genes, suggesting the presence of multiple OSC genes in plant genomes more generally. It has subsequently been established that the 13 A. thaliana OSCs all make different triterpenes (22, 25, 27, 46, 52, 66–68, 72, 73, 77, 87, 126, 132, 135, 141, 161, 162) (Figure 3). Genome mining has similarly revealed

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the presence of 12 OSC genes in the rice genome (53). Of these, 1 encodes cycloartenol synthase (56); a further 7 are predicted to encode functional triterpene synthases (53), of which 3 have been functionally characterized and shown to make parkeol (OsPS), isoarborinol (OsIAS), and achilleol and a variety of other triterpenes (OsOSC8), respectively (56, 164). The functions of the other predicted rice triterpene synthases are unknown. From the genome sequences of these two species alone, it is clear that there is likely to be a huge and as yet unrealized capability for synthesis of diverse triterpene scaffolds hidden away in plant genomes. Further progress in unveiling this capability will inevitably be aided by the extension of new genomics technologies into more plant species and accessions.

Oxidosqualene Cyclase Functional Analysis As indicated above, the primary routes for the discovery of new OSCs rely on expression-based approaches and increasingly, where genome sequence information is available, on genome mining (Figure 9). Once OSC sequences have been identified, they need to be functionally characterized. This is normally achieved through expression in yeast using strains that have been modified specifically for this purpose, either by engineering for elevated precursor supply or by using sterol auxotrophs that accumulate high levels of 2,3-oxidosqualene (6, 88, 99, 119). However, transient expression in leaves of the tobacco relative Nicotiana benthamiana using cowpea mosaic virus– based HyperTrans (CPMV-HT ) technology has recently been shown to be an effective alternative means of expressing plant OSCs (32, 69). CPMV-HT has previously proved highly effective for the rapid, transient expression of a variety of structural proteins, including antibodies, vaccines, and empty viral particles (113–116, 122). In this system, the sequence to be expressed is inserted between a modified 5 untranslated region and the 3 untranslated region of CPMV RNA-2 in the T-DNA region of one of the series of Agrobacterium tumefaciens pEAQ vectors (116), which also encode the P19 suppressor of gene silencing. This method, which involves simply infiltrating N. benthamiana leaves with A. tumefaciens containing the appropriate expression constructs using a syringe without a needle, is very quick, and results can be obtained within six days. There is no requirement for engineering the earlier steps in the isoprenoid pathway in order to achieve expression of a heterologous OSC (oat β-amyrin synthase) in N. benthamiana leaves using this system, indicating that heterologous expression of this OSC can “pull through” the necessary precursors from generic host metabolism (32).

Identiﬁcation of Tailoring Enzymes

Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org Expression of OSCs in either yeast or N. benthamiana enables the corresponding triterpene scaffold(s) to be established using mass spectrometry and (where necessary) NMR spectrometry. These

Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. triterpene scaffolds may exist in unmodiﬁed form in plants (for example, in surface waxes) (11–13, 35, 58, 78, 142). Alternatively, they may be modiﬁed by tailoring enzymes. Expression-based approaches have enabled the discovery of genes encoding candidate tailoring enzymes (5, 18, 30, 31, 37, 38, 49, 79, 94, 127, 128, 131). This is best achieved using the characterized OSC gene of interest as bait to search for other coexpressed genes with predicted functions in specialized metabolism (e.g., predicted P450 and glycosyltransferase genes). Functional validation of candidate tailoring enzymes is normally carried out in yeast. An unexpected discovery has been that the genes for several specialized metabolic pathways, including triterpene pathways, are organized in clusters in plant genomes, a form of organization that is somewhat reminiscent of the natural product biosynthetic pathway gene clusters found in Strep- tomyces and some other bacteria (100). So far, such biosynthetic gene clusters have been reported

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OSC discovery Discover new OSC sequences -/+ methyl jasmonate Genome mining Transcriptomics

Identify OSC triterpene function scaffold

Expression in yeast Transient expression in Nicotiana benthamiana

Find candidate Leaf Seed Flower Stem Lateral root Root tip Root cap Tailoring tailoring enzymes OSC enzymes OSC CYPX CYPY CYPZ Mine for linked genes Mine for coexpressed genes

Validate by coexpression in Pathway yeast or Nicotiana benthamiana validation and/or by functional analysis in plant of origin

OSC CYP1 CYP2 ... Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org Figure 9 Strategies for discovering new oxidosqualene cyclases (OSCs) and triterpene biosynthetic pathways. The images of yeast and Nicotiana Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. benthamiana leaf inﬁltration were kindly provided by the UK National Collection of Yeast Cultures and George Lomonossoff ( John Innes Centre), respectively.

for diverse classes of plant natural products from different species, including both monocots and dicots (54, 63, 69, 157). The first of the plant triterpene biosynthetic clusters to be identified was the avenacin cluster from oat (103, 107), which contains a β-amyrin synthase gene, AsbAS1, along with genes for the tailoring enzymes CYP51H10 (a P450), AsSCPL1 (a serine carboxy-peptidase- like acyltransferase), AsMT1 (a methyltransferase), and AsUGT74H5 (a glucosyltransferase) (32, 40, 89, 92, 102, 107, 108). This cluster was first defined using a classical genetics approach that involved exploiting the unusual fluorescent properties of the major oat root avenacin to screen for

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avenacin-deficient mutants of diploid oat (103), and was subsequently characterized by building a bacterial artificial chromosome contig spanning AsbAS1 (89, 92, 102, 107, 108). Other uncharacterized genes that are required for avenacin synthesis have been defined by mutation and are also linked (93, 103). The avenacin cluster spans more than 200 kb. Osbourn and coworkers (28, 29) subsequently discovered two other triterpene biosynthetic clusters in A. thaliana by mining the genome for candidate biosynthetic gene clusters that contained OSC genes, and predicted and functionally validated the thalianol and marneral clusters (35 and 38 kb, respectively). An OSC gene and two linked, functionally associated P450 genes have also recently been identified in Lotus japonicus (69). How common such clusters are in plant genomes remains to be established. Other new triterpene biosynthetic gene clusters will likely emerge as we learn more about the genomic context of OSC genes. For example, a candidate cluster consisting of an OSC gene, an acyltransferase gene, and two P450 genes, all of which are immediately adjacent to one another and coexpressed, is present in the cucumber genome. This cluster may be required for cucurbitacin synthesis (50). The genes within these biosynthetic clusters are more closely related to plant genes than to microbial ones, and they are unlikely to have been introduced by horizontal gene transfer from microbes (63). Indeed, the evidence suggests that these clusters form in dynamic regions of chromosomes, presumably in response to strong selection (28). Clustering enables coinheritance of beneficial combinations of alleles that together confer a selective advantage (e.g., the production of defense compounds), and by enabling the pathway to be inherited intact, it may also ensure that bioactive/deleterious pathway intermediates are not unleashed. It further provides for regulation at the chromatin level, for which there is a growing body of evidence. We speculate that gene clustering is a feature of specialized metabolic pathways that have evolved relatively recently in evolutionary time. Comparative genomics is now beginning to provide the first insights into the dynamics of cluster formation (28). Thus, the genes for triterpene biosynthetic pathways are coexpressed in particular plant parts, at certain growth stages, and/or in response to environmental triggers. Gene expression analysis provides a highly effective means of identifying genes for new OSCs as well as coexpressed genes for candidate tailoring enzymes. This strategy is independent of any knowledge of genomic context. However, if genome sequence is available, either from genome sequencing projects or from bacterial artificial chromosome contigs spanning OSC genes of interest, then positional information may prove to be extremely valuable for identifying gene clusters for entire triterpene biosynthetic pathways. Functional validation of new triterpene biosynthetic pathways can be demonstrated by coexpression of the OSC and candidate tailoring enzymes in yeast and/or N. benthamiana. The latter has enabled the expression of all five genes in the avenacin cluster and so has undergone proof of con-

Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org cept for expression of OSCs, P450s, acyltransferases, methyltransferases, and glycosyltransferases (32, 89, 92). There are some caveats associated with determining the biochemical functions of en-

Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. zymes based on expression in heterologous hosts because this may give misleading results. Where possible, tests of function in the plant of origin using a combination of gene knockouts, RNA interference, and/or overexpression provide a complementary means of establishing the roles of enzymes, identifying pathway intermediates, and evaluating the likely biological/ecological signiﬁ- cance of triterpene biosynthetic pathways (e.g., 18, 28, 29, 32, 40, 69, 89, 92, 93, 103, 107, 108, 123).

CONCLUSIONS Until recently, the study of triterpenes has been hindered by the intractability of these molecules to chemical synthesis and by the dearth of enzymes available for bioengineering. Recent advances in our understanding of the genes and enzymes required for triterpene synthesis are now opening

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up the potential to rapidly access these biologically and commercially important compounds. Few hypotheses currently exist regarding links between structure and activity for these diverse and complex molecules. To truly realize the scope of the metabolic diversity represented within the triterpene family, systematic studies will be needed. The development of platforms for synthetic biology–based generation and functionalization of triterpene scaffolds will enable the production of known and novel triterpenes and systematic comparison of the biological and chemical properties of these molecules. Some of the most important insights into triterpene structure and function are likely to come from investigations of the roles of these compounds in their plants of origin. So far, investigations of this kind have been limited. Knowledge of the expression profiles of different types of OSCs will guide investigations of biochemical and biological function (Figure 3, Table 1). Simple triterpenes are known to accumulate in the epicuticular and intracuticular wax layers of stem and leaf surfaces and may have physiological roles in protection against dehydration and possibly against herbivores (11–13, 35, 58, 78, 142, 166). Lupeol synthases are also expressed preferentially in root nodules of certain legumes (23, 41, 57), and lupeol is implicated in the regulation of nodule development (23). The avenacin triterpene glycosides protect oat roots against attack by soil-borne fungal pathogens such as “take-all,” which causes major yield losses on wheat (103), and other β-amyrin-derived triterpene glycosides confer resistance to flea beetle (Phyllotreta nemorum)inBarbarea vulgaris,a member of the Brassicaceae family (74). Evidence is also emerging to suggest that triterpenes may have roles in plant growth and development (28, 29, 33). The discovery of regulators of triterpene biosynthesis will represent a further important area of future research because pathway-specific transcription factors have not yet been reported.

SUMMARY POINTS 1. Triterpenes are one of the most numerous and diverse groups of plant natural products and have a wide range of applications in the food, cosmetics, pharmaceutical, and industrial biotechnology sectors. 2. Triterpenes are complex molecules that are for the most part beyond the reach of chemical synthesis. 3. Triterpenes are synthesized from 2,3-oxidosqualene by oxidosqualene cyclases (OSCs), which collectively generate more than 100 different triterpene scaffolds; these scaffolds are then further modiﬁed by tailoring enzymes (e.g., cytochrome P450s, sugar transferases, and acyltransferases) to give even greater metabolic diversity.

Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org 4. The genes for triterpene biosynthetic pathways are—in at least some cases—organized in plant genomes as coexpressed metabolic gene clusters.

Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. 5. Major advances in our understanding of the genes, enzymes, and pathways required for the synthesis of these molecules are now opening up unprecedented opportunities for triterpene metabolic engineering. 6. Suitable heterologous systems for (re)construction of triterpene biosynthetic pathways are expression in yeast and Agrobacterium-mediated transient expression in Nicotiana benthamiana leaves. 7. The development of platforms for synthetic biology–based generation and functionalization of triterpene scaffolds will enable the generation of triterpene libraries and allow systematic comparison of the biological and chemical properties of these molecules.

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DISCLOSURE STATEMENT A.O. is a coinventor on patents filed on the five cloned avenacin genes. The authors are not aware of any other affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS A.O.’s laboratory is funded by awards from the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/K005952/1), the Engineering and Physical Sciences Research Coun- cil (EP/K034359/1), and the European Union (KBBE-2013-7; TriForC); a BBSRC Institute Strategic Program Grant (“Understanding and Exploiting Plant and Microbial Metabolism,” BB/J004561/1); and the John Innes Foundation. P.O.’s laboratory is funded by an award from the BBSRC (BB/K003690/1) and BBSRC Institute Strategic Program Grants (BB/J004561/1 and “Food and Health,” BB/I015345/1). R.T. is funded by a European Commission Marie Curie International Incoming Fellowship (IIF-275689: TRICYCLE).

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Annual Review of Plant Biology Contents Volume 66, 2015

From the Concept of Totipotency to Biofortiﬁed Cereals Ingo Potrykus ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp1 The Structure of Photosystem II and the Mechanism of Water Oxidation in Photosynthesis Jian-Ren Shen ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp23 The Plastid Terminal Oxidase: Its Elusive Function Points to Multiple Contributions to Plastid Physiology Wojciech J. Nawrocki, Nicolas J. Tourasse, Antoine Taly, Fabrice Rappaport, and Francis-Andr´e Wollman pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp49 Protein Maturation and Proteolysis in Plant Plastids, Mitochondria, and Peroxisomes Klaas J. van Wijk ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp75 United in Diversity: Mechanosensitive Ion Channels in Plants Eric S. Hamilton, Angela M. Schlegel, and Elizabeth S. Haswell pppppppppppppppppppppppp113 The Evolution of Plant Secretory Structures and Emergence of Terpenoid Chemical Diversity Bernd Markus Lange pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp139 Strigolactones, a Novel Carotenoid-Derived Plant Hormone Salim Al-Babili and Harro J. Bouwmeester ppppppppppppppppppppppppppppppppppppppppppppppp161 Moving Toward a Comprehensive Map of Central Plant Metabolism Ronan Sulpice and Peter C. McKeown ppppppppppppppppppppppppppppppppppppppppppppppppppppp187 Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org Engineering Plastid Genomes: Methods, Tools, and Applications in

Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. Basic Research and Biotechnology Ralph Bock ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp211 RNA-Directed DNA Methylation: The Evolution of a Complex Epigenetic Pathway in Flowering Plants Marjori A. Matzke, Tatsuo Kanno, and Antonius J.M. Matzke ppppppppppppppppppppppppp243 The Polycomb Group Protein Regulatory Network Iva Mozgova and Lars Hennig ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp269

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The Molecular Biology of Meiosis in Plants Rapha¨el Mercier, Christine M´ezard, Eric Jenczewski, Nicolas Macaisne, and Mathilde Grelon ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp297 Genome Evolution in Maize: From Genomes Back to Genes James C. Schnable ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp329 Oxygen Sensing and Signaling Joost T. van Dongen and Francesco Licausi pppppppppppppppppppppppppppppppppppppppppppppppp345 Diverse Stomatal Signaling and the Signal Integration Mechanism Yoshiyuki Murata, Izumi C. Mori, and Shintaro Munemasa pppppppppppppppppppppppppppp369 The Mechanism and Key Molecules Involved in Pollen Tube Guidance Tetsuya Higashiyama and Hidenori Takeuchi ppppppppppppppppppppppppppppppppppppppppppppp393 Signaling to Actin Stochastic Dynamics Jiejie Li, Laurent Blanchoin, and Christopher J. Staiger ppppppppppppppppppppppppppppppppp415 Photoperiodic Flowering: Time Measurement Mechanisms in Leaves Young Hun Song, Jae Sung Shim, Hannah A. Kinmonth-Schultz, and Takato Imaizumi pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp441 Brachypodium distachyon and Setaria viridis: Model Genetic Systems for the Grasses Thomas P. Brutnell, Jeffrey L. Bennetzen, and John P. Vogel ppppppppppppppppppppppppppp465 Effector-Triggered Immunity: From Pathogen Perception to Robust Defense Haitao Cui, Kenichi Tsuda, and Jane E. Parker pppppppppppppppppppppppppppppppppppppppppp487 Fungal Effectors and Plant Susceptibility Libera Lo Presti, Daniel Lanver, Gabriel Schweizer, Shigeyuki Tanaka, Liang Liang, Marie Tollot, Alga Zuccaro, Stefanie Reissmann, and Regine Kahmann pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp513 Responses of Temperate Forest Productivity to Insect and Pathogen Disturbances Annu. Rev. Plant Biol. 2014.65:225-257. Downloaded from www.annualreviews.org Charles E. Flower and Miquel A. Gonzalez-Meler ppppppppppppppppppppppppppppppppppppppp547

Access provided by Guangzhou University of Chinese Medicine on 03/10/17. For personal use only. Plant Adaptation to Acid Soils: The Molecular Basis for Crop Aluminum Resistance Leon V. Kochian, Miguel A. Pi˜neros, Jiping Liu, and Jurandir V. Magalhaes ppppppppp571 Terrestrial Ecosystems in a Changing Environment: A Dominant Role for Water Carl J. Bernacchi and Andy VanLoocke pppppppppppppppppppppppppppppppppppppppppppppppppppp599

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