Can Arabidopsis make complex alkaloids?

Opinion

TRENDS in Plant Science

Vol.9 No.3 March 2004

Can Arabidopsis make complex alkaloids? Peter J. Facchini1, David A. Bird1 and Benoit St-Pierre2 1

Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4 Laboratoire de Physiologie Ve´ge´tale, EA2106 ‘Biomole´cules et Biotechnologies Ve´ge´tale’, UFR Sciences et Techniques, Parc de Grandmont, Universite´ de Tours, 37200 Tours, France 2

Alkaloids are a large group of secondary metabolites with diverse biosynthetic origins and limited taxonomic distribution. They are generally defined by the occurrence of a nitrogen atom in an oxidative state within a heterocyclic ring. Homologues for many enzymes involved in alkaloid biosynthesis have been detected in the Arabidopsis genome, in spite of the apparent lack of complex alkaloids in this plant. The most common homologues are putative genes encoding decorative enzymes such as hydroxylases, methyltransferases and acetyltransferases. Nevertheless, the recent discovery that Arabidopsis produces volatile terpenoids, coupled with the multitude of alkaloid biosynthetic gene homologues, lends credibility to the suggestion that undiscovered alkaloids are also present in this plant. Alkaloids are a large, diverse group of natural products found in , 20% of plant species. They are generally defined by the occurrence of a nitrogen atom in an oxidative state within a heterocyclic ring. Many of the , 12,000 known alkaloids produced by plants display potent pharmacological activities and several are widely used as pharmaceuticals (Figure 1). As secondary metabolites, alkaloids are derived from the products of primary metabolism, with amino acids serving as their main precursors [1]. However, unlike most other groups of natural products, the many structural types of alkaloids have independent biosynthetic origins. For example, isoquinoline alkaloids such as morphine and berberine are synthesized from tyrosine, indole alkaloids such as vinblastine are derived from tryptophan, and tropane alkaloids such as cocaine and scopolamine are produced from ornithine. Alkaloid biosynthetic pathways are typically composed of multiple catalytic steps that not only form a basic structural nucleus but also modify nascent alkaloid molecules through various carbon-ring modifications and a multitude of decorative reactions including hydroxylations, methylations, acetylations and glycosylations [2]. Thus, it is not surprising that many different enzyme types are involved in plant alkaloid metabolism [1]. Many of the same functional categories of enzymes are involved in other natural product and primary metabolic pathways. In recent years, much progress has been made on the isolation of genes encoding enzymes involved in the Corresponding author: Peter J. Facchini ([email protected]).

biosynthesis of several different alkaloids, including those of the isoquinoline, indole, tropane and purine classes [1]. Notable advances include the identification of most genes involved in berberine production (Figure 2), the cloning of several genes required for the formation of the terpenoid indole alkaloid vindoline (Figure 3) and the isolation of genes encoding key steps in the biosynthesis of scopolamine and the purine alkaloid caffeine [1]. The nearly complete collection of molecular clones encoding enzymes involved in berberine biosynthesis provides an ideal model pathway to survey the diversity of enzymes typically involved in alkaloid formation. These include three O-methyltransferases [3,4], an N-methyltransferase [5], two P450-dependent monooxygenases [6,7], a pyridoxylphosphate-dependent aromatic amino acid decarboxylase [8], a FAD-dependent oxidoreductase [9] and norcoclaurine synthase (NCS), a novel enzyme catalysing the first committed step of the pathway (N. Samanani and P. Facchini, unpublished). Arabidopsis accumulates the simple alkaloid camalexin as a phytoalexin [10]. However, completion of the Arabidopsis genome sequence has shown that genes involved in the biosynthesis of complex alkaloids (Figure 1) in exotic plant species such as opium poppy (Papaver somniferum), Madagascar periwinkle (Catharanthus roseus) and deadly nightshade (Atropa belladonna) are represented by multiple homologues in Arabidopsis. In spite of the apparent lack of complex alkaloid accumulation in Arabidopsis, the presence of alkaloid biosynthetic gene homologues has sometimes been interpreted as an indication that alkaloids might be produced in this plant either at low levels or under certain environmental conditions. In this article, we consider the possibility that Arabidopsis has the capacity to synthesize and accumulate complex alkaloids. Although support for and evidence against this suggestion are discussed, our main objective is to provoke some rational thought about the perplexing number of sequences in the Arabidopsis genome with homology to the ever-increasing collection of alkaloid biosynthetic genes. Multitude of alkaloid biosynthetic gene homologues in Arabidopsis In the original paper describing the complete Arabidopsis genome sequence, members of the Arabidopsis Genome Initiative concluded that ‘the presence of 12 genes with

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Opinion

TRENDS in Plant Science

HO

O N+ O

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HO Morphine

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HO C2H5 N N H

H

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CO2CH 3

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H3C HO CO2CH 3 Vinblastine

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CH 3 N Nicotine TRENDS in Plant Science

Figure 1. Some pharmaceutically and socially important alkaloids derived from plants. Morphine (a narcotic analgesic) and berberine (an antimicrobial) are examples of benzylisoquinoline alkaloids. The antineoplastic agent vinblastine is a dimeric terpenoid indole alkaloid, whereas cocaine (a topical anaesthetic) and scopolamine (which is used to treat motion sickness) are representative of the tropane alkaloids. The stimulant caffeine is a purine alkaloid, whereas nicotine (a pyrrolidine alkaloid) is a psychostimulant.

sequence similarity to berberine bridge enzyme [BBE], and 13 genes with similarity to tropinone reductase [TRI], suggests that Arabidopsis might have the ability to produce alkaloids’ [11]. BBE and TRI are key branchpoint enzymes in the biosynthesis of certain isoquinoline and tropane alkaloids, respectively. A postulated capacity for complex alkaloid biosynthesis was also suggested following the detection of stress-inducible, putative BBE and tyrosine transaminase genes in Arabidopsis [12], and www.sciencedirect.com

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the identification of a plant efflux carrier with the ability to transport berberine and related alkaloids [13]. BBE belongs to the general FAD-dependent oxidoreductase family [14] and TRI to the short-chain, non-metal dehydrogenase family [15]. Although BBE and TRI catalyse reactions specific to alkaloid biosynthesis, other members of these families would be expected to act on a range of substrates, as recently demonstrated by the functional characterization of a tobacco BBE sequence homologue as a glucose oxidase [44]. The isolation of a berberine transporter might also appear as strong support for the occurrence of complex alkaloids in Arabidopsis. However, the common acceptance of berberine as a substrate for such ATP binding cassette (ABC) carriers [16] suggests that even the ABC transporter identified in Japanese goldenthread (Coptis japonica), a berberineproducing plant, is not necessarily involved in berberine metabolism [17]. An important undercurrent of the current debate is the well known, yet often overlooked, tenet that sequence homology is an insufficient datum on which to base predictions of specific enzyme function, much less the overall biosynthetic capability of a plant. Examples of enzymes, particularly those involved in natural product metabolism, with marginal, or even single, amino acid substitutions that display absolutely distinct substrate specificities abound in plants [18,19]. Part of the problem is related to the protocol used to annotate genomic and expressed sequence tag data. Such annotations are intended only to identify conserved sequence domains and to suggest putative functions as a guide to empirical characterization. Terpenoids were only recently discovered in Arabidopsis The suggestion that Arabidopsis can produce unexpected natural products is not without precedent: the capacity for complex alkaloid biosynthesis might seem a logical conjecture from the recent discovery that Arabidopsis flowers emit volatile terpenoids [20]. The first indication that Arabidopsis has the potential for terpenoid biosynthesis came via the same resource – its genome sequence – that is provoking suggestions of complex alkaloid production. More than 30 genes with sequences homologous to known terpene synthases (TPSs) are predicted in the Arabidopsis genome, a few of which have been functionally characterized [21]. However, TPSs are a unique class of enzymes, all of which catalyse the conversion of allylic prenyl diphosphate intermediates to a range of terpenoids. Most of the enzymes involved in complex alkaloid biosynthesis for which sequence homologues have been detected in the Arabidopsis genome belong to broad functional categories including O-methyltransferases [22], O-acetyltransferases [23], P450-dependent monooxygenases [24], FAD-dependent oxidoreductases [44] and 2-oxoglutarate-dependent dioxygenases [25]. Members of several of these enzyme families have been shown to catalyse reactions that involve a diverse range of substrates including, but not restricted to, alkaloids. O-Methyltransferases, for example, are involved in the synthesis of virtually every type of secondary metabolite produced in plants [26]. The discovery of TPS genes in

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TRENDS in Plant Science

CHO HO

HO

4-Hydroxylphenylacetaldehyde

NH

HO

NCS

+

H

HO HO NH2

HO

(S)-Norcoclaurine

Dopamine 6OMT TYDC H3CO COOH

HO

NH

HO

H

NH2

HO

L-Dopa

HO

(S)-Coclaurine CNMT H3CO

H3CO NCH 3

HO

CYP80B1

H

HO

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HO

H

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(S)-3′-HydroxyN-methylcoclaurine

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H

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N H OH

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(S)-Scoulerine SOMT

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N O

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(S)-Canadine

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(S)-Tetrahydrocolumbamine

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Vol.9 No.3 March 2004

Arabidopsis suggests the existence of terpenoid biosynthesis because TPSs are involved in the formation of the basic structural nucleus that defines this class of natural products. By contrast, the identification of modifying enzymes that catalyse methylations, acetylations, hydroxylations and even carbon-ring rearrangements do not inherently suggest the occurrence of complex alkaloids because of the diverse nature of the substrates and reaction products associated with such enzymes. Indeed, many such enzymes also participate in the biosynthesis of complex terpenoids after the initial TPS-catalysed reactions [27]. Entry-point enzymes and substrate availability Unlike TPS, key entry-point enzymes that catalyse the formation of basic alkaloid nuclei have not been functionally identified in Arabidopsis, and in most cases, the molecular cloning of such enzymes has not been achieved. However, two cases might be used to fuel the debate – NCS, which catalyses the formation of the central precursors in the biosynthesis of all benzylisoquinoline alkaloids (Figure 2), and strictosidine synthase (STR), which catalyses the formation of the central precursors in the biosynthesis of monoterpenoid indole alkaloids (Figure3) [1]. The recent cloning of NCS from meadow rue (Thalictrum flavum), another berberine-producing plant, has shown that the nearest homologues in Arabidopsis are a family of genes exhibiting only 30 – 35% sequence identity with NCS (N. Samanani and P. Facchini, unpublished). Homologues for STR also occur in the Arabidopsis genome but, again, the most related sequences are a family of genes exhibiting only 25 – 35% identity with STR. The limited similarity between NCS, or STR, and their Arabidopsis sequence homologues contrasts with the extensive identity between other groups of alkaloid biosynthetic genes and their counterparts in the Arabidopsis genome. Sequence identities of 50 –70% are not uncommon when comparing the decarboxylases, methyltransferases, FAD-dependent oxidoreductases and P450dependent monooxygenases involved in the biosynthesis of berberine with sequence homologues in Arabidopsis (Figure 2). Similar identities are found between some P450-dependent monooxygenases or 2-oxoglutaratedependent dioxygenases predicted in the Arabidopsis genome, and functional homologues involved in the biosynthesis of the monoterpenoid indole alkaloid vindoline (Figure 3). However, the functional identity of such sequence homologues must be tested experimentally before any real conclusions can be drawn. There are reasons to suspect that NCS and STR homologues in Arabidopsis do not catalyse the formation of norcoclaurine and strictosidine, respectively. First, NCS shares homology with pathogenesis-related-(PR)10

OCH3

Berberine TRENDS in Plant Science

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Figure 2. Biosynthetic pathway leading to berberine showing reactions catalysed by enzymes for which corresponding cDNAs have been isolated. Abbreviations: 6OMT, norcoclaurine-6-O-methyltransferase; CNMT, coclaurine N-methyltransferase; CYP719, canadine synthase; CYP80B1, (S)-N-methylcoclaurine-30 -hydroxylase; 40 OMT, 30 -hydroxy-N-methylcoclaurine-40 -O-methyltransferase; BBE, berberine bridge enzyme; NCS, norcoclaurine synthase; SOMT, (S)-scoulerine-9-O-methyltransferase; TYDC, tyrosine/dopa decarboxylase.

Opinion

TRENDS in Plant Science

G10H CH2OH

CH2OH CH2OH

Geraniol

10-Hydroxygeraniol

HO HO COOH

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NH2

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NH

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OH

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H3CO2C

Strictosidine aglycoside

Tabersonine T16H

N

N

H HO

N H

H H3CO

N H3C HO

CO2CH3

16-Hydroxytabersonine

CO2CH3

Desacetoxyvindoline D4H

N

N

DAT

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H H3CO

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OCH2CH3

H3C HO CO2CH3

Vindoline

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proteins, which are ubiquitous in plants (N. Samanani and P. Facchini, unpublished). Moreover, STR sequence homologues are found in plants, bacteria, insects and even mammals. This ancient group of enzymes is defined by a common hydrolyase activity, a reverse reaction acting on a carbon–nitrogen bond. For example, bacterial STR homologues have been identified as gluconolactonase (http://www.ncbi.nlm.nih.gov/sutils/blink.cgi?pid ¼ 6664319), which catalyses a hydrolyase reaction. The ability of a plant species to synthesize a specific alkaloid depends on the presence of a key entry-point enzyme, such as NCS or STR, so a key question is whether Arabidopsis has such enzymes. It is also important to consider that complex alkaloids would not be produced in Arabidopsis even if entry-point enzymes, such as STR and NCS, were present unless their substrates were also available. The biosynthesis of these substrates also involves multiple, specialized enzymes, such as geraniol 10-hydroxylase [28] and secologanin synthase [29], which are involved in the formation of the iridoid substrate secologanin (Figure 3). Adaptive and exaptive evolution of secondary metabolism The question of whether or not Arabidopsis has the ability to synthesize complex alkaloids is really a discussion about the evolution of plant secondary metabolism. This subject has been treated from two main perspectives – the recruitment of natural product enzymes from populations of duplicated genes in plant genomes [22,30,31] and the cost – benefit balance of acquiring the ability to produce specific products [32,33]. When higher plants are analysed at the phytochemical level, the limited taxonomic distribution of specific alkaloids becomes apparent (Figure 4). Two possibilities might explain the lack of all alkaloid types in every plant taxon – either most, or all, alkaloid types were synthesized in ancestral plants but many were subsequently lost in their descendents, or some alkaloid types are recent products of the continuous evolution of secondary metabolism. Certainly, a combination of both events might have contributed to the modern distribution of alkaloids (and other natural products) found in plants. It is intriguing that the isoquinoline alkaloids are common in more ancient families, such as the Magnoliaceae, Ranunculaceae and Papaveraceae, but are also found sporadically in several other taxa (Figure 4). By contrast, indole, tropane and quinolizidine alkaloids are restricted to arguably less primitive taxa (Figure 4). Perhaps Arabidopsis displays a genomic footprint for complex alkaloid biosynthesis but the substrate specificity of some, if not all, of the gene products has changed. However, it is important to remember that the ability to synthesize and accumulate any given natural product, including cytotoxic alkaloids, is dependent on more than the mere presence of a collection of appropriately functional enzymes. The involvement of specific transcriptional regulators orchestrating the

Deacetylvindoline TRENDS in Plant Science

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Vol.9 No.3 March 2004

Figure 3. Biosynthetic pathway leading to vindoline showing reactions catalysed by enzymes for which corresponding cDNAs have been isolated. Abbreviations: D4H, desacetoxyvindoline 4-hydroxylase; DAT, deacetylvindoline 4-O-acetyltransferase; SGD, strictosidine b-D -glucosidase; STR, strictosidine synthase; T16H, tabersonine 16-hydroxylase; TDC, tryptophan decarboxylase.

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Pyrrolizidine (ornithine) Pyrrolidine (ornithine)

TRENDS in Plant Science

Vol.9 No.3 March 2004

Quinolizidine (lysine)

Isoquinoline (tyrosine)

Tropane (ornithine)

Piperidine (lysine)

Purine (adenine) Indole (tryptophan)

Figure 4. Distribution of selected alkaloids in the angiosperms. Adapted, with permission, from Ref. [43], the figure depicts a phylogenetic tree in transection, with each order represented by a different branch. The thickness of the branch is roughly proportional to the number of species in the order, with the relative position of the orders defined through a consideration of multiple parameters. The distribution of eight major classes of alkaloids is shown. The extent to which an alkaloid type is found in each order is represented as follows: small dots, one to three genera or isolated cases; medium dots, up to 10% of the genera; large dots, up to 25% of the genera; entirely filled, . 25% of the genera.

expression of relevant genes [34], the cellular and subcellular localization of enzymes, and the inter- and intracellular transport of pathway intermediates and products [35–38] are all crucial factors in the capacity of a plant to produce complex alkaloids and other natural products. Although it is difficult to envisage the multitude and complexity of evolutionary events required for the establishment of a new metabolic pathway, it is simpler to imagine the loss of an existing pathway as the result of even a single gene mutation in, for example, an entry-point enzyme or transcription factor. What evolutionary mechanism can explain the advantage of losing the ability to synthesize certain types of secondary metabolites? If we contend that individual alkaloids, and other natural products, confer selective and environment-specific advantages, it is difficult to explain the benefit of losing metabolites. However, if we consider that the selective advantage of secondary metabolism might be based primarily on the ability of a plant to produce a diverse and dynamic spectrum of natural products, which might be used as a repository of chemical defences against continuously changing environmental challenges, the lost capacity to synthesize a specific type of www.sciencedirect.com

alkaloid might be negligible. Perhaps plant secondary metabolism evolves, at least in part, along the EXAPTIVE lines (see Glossary) proposed by Gould and Lewontin [39] to explain the appearance of complex traits whose Glossary Adaptionist: adaptive evolution is the classic Darwinian theory of evolution based on the retention of genetic traits over generations that confer a selective advantage to certain members of a population with respect to specific environmental challenges. Bet-hedging: the retention of multiple traits designed to allow the flexible adaptation to changing environmental challenges, the success of which depends on the effectiveness of at least one strategy. A popular example is the observed variation in the timing of seed germination to promote the survival of at least some progeny. Exaptive: exaptive evolution is an alternative evolutionary theory suggesting that some functional characteristics might have arisen as a result of indirect environmental pressures, rather than progressive adaptations to perceived challenges via natural selection. For example, feathers probably evolved to assist in thermoregulation but were subsequently advantageous for flight [42]. Metabolic plasticity: the ability of a plant species efficiently to alter its profile of metabolites in response to frequently changing environmental conditions. Spandrels: this word originates from a unique architectural feature of the San Marco Cathedral in Venice, and is used metaphorically to describe biological characteristics that arise as evolutionary byproducts rather than by direct adaptations. These byproducts might later become functional through exaptation.

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intermediate stages do not logically confer a selective advantage. Plants maintain a broad array of redundant genes whose main purpose might be to provide the ‘SPANDRELS ’ necessary to build a complex, ever-changing arsenal of natural products required for survival. Could secondary metabolism be the ultimate form of evolutionary ‘BET-HEDGING ’ in plants [40]? ADAPTIONIST thinking has dominated theories about the evolution of secondary metabolism. That is, natural products are thought to exist because they are useful to the plant [22]. Exaptive theory recognizes that some evolutionary events result from genetic drift not necessarily (or, at least, immediately) coupled to selective adaptations. The spandrels of San Marco in Venice were used as the classic analogy for exaptive evolution – specifically, that forms and spaces might arise as the result of another decision in design [40]. Perhaps METABOLIC PLASTICITY is the main driving force behind the evolution of natural product metabolism. A selective advantage is based as much, or perhaps more, on a plant’s ability to maintain a diverse collection of potentially bioactive molecules and continuously to shuffle the natural product deck, as it is on plants stumbling upon the ability to make individual products that confer protection against specific, albeit temporary (in an evolutionary sense), environmental challenges. The estimate that 15 – 20% of the . 25,000 genes in Arabidopsis are involved in natural product biosynthesis supports [41] the contention that variation and plasticity are the selectable traits that power the evolution of secondary metabolites. Arabidopsis appears to have many more genes than necessary to produce all the natural products so far identified in this plant. A combination of exaptive and adaptive mechanisms would most comfortably explain existing alkaloid profiles in plants. Evidence to support the selection of individual alkaloids can be gleaned from the widespread occurrence of antimicrobial isoquinoline alkaloids, such as berberine, throughout the Papaverales and Ranunculales. The highly restricted distribution of the related alkaloid morphine supports the ancient evolution and sustained selection of the berberine pathway compared with the more recent appearance and/or limited presentation of other, currently less efficacious, alkaloids. Extensive metabolic profiling projects are being conducted with Arabidopsis exposed to various environmental conditions, and this work should ultimately provide evidence on the numbers and identity of secondary metabolites present in this plant. An answer to the question of whether or not Arabidopsis, or any other plant, has the capacity to synthesize complex alkaloids might also result from the detailed functional characterization of every gene product. This will be difficult. It will also be hard to prove that exaptation is a relevant concept in the evolution of plant secondary metabolism. However, we hope that our attempt to introduce an alternative perspective will lead to increased debate and reflection on the evolution of secondary metabolism. We have also striven to provide some food for thought about the utility of genome and expressed sequence tag annotations, particularly in the context of plant alkaloid and, more generally, natural product metabolism. www.sciencedirect.com

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34 Memelink, J. et al. (2001) ORCAnization of jasmonate-responsive gene expression in alkaloid metabolism. Trends Plant Sci. 6, 212 – 219 35 St-Pierre, B. et al. (1999) Multicellular compartmentation of Catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediate. Plant Cell 11, 887 – 900 36 De Luca, V. and St-Pierre, B. (2000) The cell and developmental biology of alkaloid biosynthesis. Trends Plant Sci. 5, 168 – 173 37 Bird, D.A. and Facchini, P.J. (2001) Berberine bridge enzyme, a key branch-point enzyme in benzylisoquinoline alkaloid biosynthesis, contains a vacuolar-sorting determinant. Planta 213, 888– 897 38 Bird, D.A. et al. (2003) A tale of three cell types: alkaloid biosynthesis is localized to sieve elements in opium poppy. Plant Cell 15, 2626 – 2635 39 Gould, S.J. and Lewontin, R.C. (1979) The spandrels of San Marco and the Panglossian paradigm: a critique of the adaptationist programme. Proc. R. Soc. London B. Biol. Sci. 205, 581– 598 40 Clauss, M.J. and Venable, D.L. (2000) Seed germination in desert annuals: an empirical test of adaptive bet hedging. Am. Nat. 155, 168– 186 41 Somerville, C. and Somerville, S. (1999) Plant functional genomics. Science 285, 380– 383 42 Gould, S.J. and Vrba, E.S. (1982) Exaptation: a missing term in the science of form. Paleobiology 8, 4 – 15 43 Dahlgren, R.M.T. (1980) A revised system of classification of the angiosperms. Bot. J. Linnean Soc. 80, 91– 124 44 Carter, C.J. and Thornburg, R.W. (2004) Tobacco nectarin V is a flavincontaining berberine bridge enzyme-like protein with glucose oxidase activity. Plant Physiol. 134, 460469

Auxin 2004 22–28 May 2004 Orthodox Academy of Crete, Kolympari, Crete, Greece Organizers: Sakis Theologis and Goran Kjell Sandberg For more information, please see http://pgec-genome.ars.usda.gov/Auxin2004/

3rd International Symposium on Plant Dormancy: From Molecular Level to the Whole Plant 24–28 May 2004 Wageningen, The Netherlands Organized by: Wageningen Seed Centre For more information, please see http://www.seedcentre.nl/

Botany 2004 Alpine Diversity: Adapted to the Peaks 31 July–5 August 2004 Snowbird Resort, Salt Lake City, UT, USA For more information, please see http://www.2004.botanyconference.org/ www.sciencedirect.com