SEARCH

SEARCH BY CITATION

Keywords:

  • benzylisoquinoline alkaloids;
  • monoterpenoid indole alkaloids;
  • Papaver somniferum;
  • Catharanthus roseus;
  • secondary metabolism;
  • non-model systems

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Benzylisoquinoline alkaloid metabolism
  5. Monoterpenoid indole alkaloid metabolism
  6. The role of ‘omics’ technologies to study plant alkaloid biosynthesis
  7. Compartmentalization and trafficking
  8. Gene regulation and signal transduction
  9. Metabolic engineering of MIA and BIA biosynthesis
  10. Outlook
  11. Acknowledgements
  12. References

Alkaloids represent a large and diverse group of compounds that are related by the occurrence of a nitrogen atom within a heterocyclic backbone. Unlike other types of secondary metabolites, the various structural categories of alkaloids are unrelated in terms of biosynthesis and evolution. Although the biology of each group is unique, common patterns have become apparent. Opium poppy (Papaver somniferum), which produces several benzylisoquinoline alkaloids, and Madagascar periwinkle (Catharanthus roseus), which accumulates an array of monoterpenoid indole alkaloids, have emerged as the premier organisms used to study plant alkaloid metabolism. The status of these species as model systems results from decades of research on the chemistry, enzymology and molecular biology responsible for the biosynthesis of valuable pharmaceutical alkaloids. Opium poppy remains the only commercial source for morphine, codeine and semi-synthetic analgesics, such as oxycodone, derived from thebaine. Catharanthus roseus is the only source for the anti-cancer drugs vinblastine and vincristine. Impressive collections of cDNAs encoding biosynthetic enzymes and regulatory proteins involved in the formation of benzylisoquinoline and monoterpenoid indole alkaloids are now available, and the rate of gene discovery has accelerated with the application of genomics. Such tools have allowed the establishment of models that describe the complex cell biology of alkaloid metabolism in these important medicinal plants. A suite of biotechnological resources, including genetic transformation protocols, has allowed the application of metabolic engineering to modify the alkaloid content of these and related species. An overview of recent progress on benzylisoquinoline and monoterpenoid indole alkaloid biosynthesis in opium poppy and C. roseus is presented.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Benzylisoquinoline alkaloid metabolism
  5. Monoterpenoid indole alkaloid metabolism
  6. The role of ‘omics’ technologies to study plant alkaloid biosynthesis
  7. Compartmentalization and trafficking
  8. Gene regulation and signal transduction
  9. Metabolic engineering of MIA and BIA biosynthesis
  10. Outlook
  11. Acknowledgements
  12. References

Alkaloids are a group of nitrogen-containing natural products that are found in approximately 20% of plant species. Many of the approximately 12 000 plant alkaloids for which structures have been elucidated exhibit potent biological activity. Unlike other types of secondary metabolites, the numerous and diverse alkaloid structural types have independent biosynthetic origins. A discussion of general alkaloid metabolism in plants reveals several interesting patterns, but specific details regarding the biochemistry, molecular and cell biology of alkaloid biosynthesis are unique to each category. Over the past several decades, two alkaloid-producing plants have emerged as model systems based on the number of characterized cDNAs encoding biosynthetic enzymes and regulatory proteins. With the establishment of cellular localization models and the application of genomics technologies, Papaver somniferum (opium poppy) and Catharanthus roseus (Madagascar periwinkle) have emerged as key species in the study of alkaloid metabolism in plants.

Opium poppy accumulates a number of benzylisoquinoline alkaloids (BIA), a term that collectively describes approximately 2500 elucidated natural product structures found mainly in the Papaveraceae, Ranunculaceae, Berberidaceae and Menispermaceae. The most prominent compounds are the narcotic analgesic morphine, the cough suppressant codeine, the muscle relaxant papaverine, and the anti-microbial agents sanguinarine and berberine. Molecular clones encoding at least 20 BIA biosynthetic enzymes have been isolated, and the pace of gene discovery will undoubtedly increase with the extensive application of new technologies. Catharanthus roseus is a valuable and important medicinal plant that produces complex anti-tumor drugs that are effective against a variety of cancers (van der Heijden et al., 2004). The monoterpenoid indole alkaloids (MIAs) are one of the largest and most diverse groups of plant secondary metabolites (approximately 3000 structures), occurring mainly in the Apocynaceae, Loganiaceae and Rubiaceae. The complex chemistry of MIAs is related to powerful biological activities, and several drugs have been developed, including reserpine for the treatment of neurological disorders, vinblastine and vincristine as anti-neoplastic agents, and yohimbine as a vasodilator.

The biosynthesis (Facchini, 2006) and metabolic engineering (Larkin and Harrigan, 2007; Sato et al., 2007) of BIAs have recently been reviewed. Several reviews have also been published on the analysis (Hisiger and Jolicoeur, 2007), chemistry (O’Connor and Maresh, 2006), biosynthesis (El-Sayed and Verpoorte, 2007; Hedhili et al., 2007; Loyola-Vargas et al., 2007; Oudin et al., 2007a), regulation (Memelink and Gantet, 2007), intra-cellular, inter-cellular and organ-specific compartmentation (Mahroug et al., 2007), plant cell culture-based production (Zhao and Verpoorte, 2007), biotechnological production (Zárate and Verpoorte, 2007), functional genomics (Goossens and Rischer, 2007) and transport (Roytrakul and Verpoorte, 2007) of MIAs in C. roseus. Together, these reviews reflect the profusion of scientific achievements that highlight opium poppy and C. roseus as two of the most thoroughly investigated medicinal plants. This review outlines recent developments involving the application of a wide array of biochemical, molecular, cellular and genomic techniques have allowed opium poppy and C. roseus to emerge as model non-model systems for the investigation of alkaloid biosynthesis in plants.

Benzylisoquinoline alkaloid metabolism

  1. Top of page
  2. Summary
  3. Introduction
  4. Benzylisoquinoline alkaloid metabolism
  5. Monoterpenoid indole alkaloid metabolism
  6. The role of ‘omics’ technologies to study plant alkaloid biosynthesis
  7. Compartmentalization and trafficking
  8. Gene regulation and signal transduction
  9. Metabolic engineering of MIA and BIA biosynthesis
  10. Outlook
  11. Acknowledgements
  12. References

Genes for the biosynthesis of simple BIAs

Fundamentally, BIA biosynthesis involves the condensation of two tyrosine derivatives, and begins with the decarboxylation of tyrosine or dihydroxyphenylalanine (DOPA) to yield tyramine and dopamine, respectively. Tyrosine decarboxylase (TYDC) is encoded by a large gene family with about 15 members in opium poppy (Facchini and De Luca, 1994). Dopamine is the precursor for the isoquinoline moiety, and 4-hydroxyphenylacetaldehyde (4-HPAA), the deamination product of tyramine, is incorporated into the benzyl component (Figure 1). Dopamine and 4-HPAA are coupled by a Pictet–Spengler condensation catalyzed by norcoclaurine synthase (NCS), the first committed step in the pathway. The enzyme was purified from Thalictrum flavum, and corresponding cDNAs have been isolated and functionally characterized from T. flavum and opium poppy (Liscombe et al., 2005; Samanani and Facchini, 2002; Samanani et al., 2004). NCS is related to the pathogenesis-related protein pathogenesis-related 10 (PR10) and Bet v 1 allergen protein families. However, homologous PR10 proteins from opium poppy and other plants are not catalytically active (Liscombe et al., 2005). Biochemical characterization of recombinant T. flavum NCS using a continuous enzyme assay based on circular dichroism spectroscopy following the generation of one enantiomer – (S)-norcoclaurine – revealed a reaction mechanism involving a two-step cyclization with a direct electrophilic aromatic substitution (Luk et al., 2007). Recently, a second enzyme that also produces only (S)-norcoclaurine, but shows sequence similarity to 2-oxoglutarate-dependent dioxygenases was isolated from Coptis japonica (Minami et al., 2007). However, this enzyme does not possess a 2-oxoglutarate-binding domain and requires ferrous ions, rather than 2-oxoglutarate or oxygen, for activity. It is remarkable that two proteins belonging to different protein families can catalyze the same reaction in vitro, and the relative participation of each enzyme in BIA biosynthesis in vivo remains to be determined.

image

Figure 1.  Biosynthesis of BIAs including morphine, sanguinarine and berberine. Enzymes for which corresponding cDNAs have been isolated are shown in red. The key branch-point intermediate (S)-reticuline is highlighted in yellow. Abbreviations: 4′OMT, 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase; 6OMT, norcoclaurine 6-O-methyltransferase; 7OMT, reticuline 7-O-methyltransferase; BBE, berberine bridge enzyme; CFS, cheilanthifoline synthase; CNMT, coclaurine N-methyltransferase; COR, codeinone reductase; Cyp719A1, canadine synthase; Cyp719A2, stylopine synthase; Cyp719B1, salutaridine synthase; Cyp80A1, berbamunine synthase; Cyp80B1, N-methylcoclaurine 3′-hydroxylase; DBOX, dihydrobenzophenanthridine oxidase; DRR, 1,2-dehydroreticuline reductase; DRS, 1,2-dehydroreticuline synthase; MSH, N-methylstylopine 14-hydroxylase; NCS, norcoclaurine synthase; P6H, protopine 6-hydroxylase; SalAT, salutaridinol 7-O-acetyltransferase; SOMT, scoulerine 9-O-methyltransferase; SalR, salutaridine:NADPH 7-oxidoreductase; STOX, (S)-tetrahydroxyprotoberberine oxidase; TNMT, tetrahydroprotoberberine cis-N-methyltransferase; TYDC, tyrosine decarboxylase.

Download figure to PowerPoint

The conversion of (S)-norcoclaurine to (S)-reticuline involves an O-methylation at position 6, an N-methylation, a 3′-hydroxylation and a second 4′-O-methylation (Figure 1). The two O-methyltransferases – norcoclaurine 6-O-methyltransferase (6OMT) and 3′-hydroxy-N-methylcoclaurine 4′-O-methytransferase (4′OMT) – display strict regiospecificity, and cDNAs for each have been obtained from opium poppy and Coptis japonica (Morishige et al., 2000; Ounaroon et al., 2003; Ziegler et al., 2005). Cognate cDNAs encoding coclaurine N-methyltransferase (CNMT) have been isolated from opium poppy and Coptis japonica and are more similar to S-adenosyl-L-methionine (SAM)-dependent cyclopropane fatty acid synthases than to other N-methyltransferases (Choi et al., 2002; Facchini and Park, 2003). The hydroxylation of N-methylcoclaurine is catalyzed by a P450 mono-oxygenase of the Cyp80B sub-family (Huang and Kutchan, 2000; Ikezawa et al., 2003; Pauli and Kutchan, 1998; Samanani et al., 2005).

Most BIA structural types are derived from the central pathway intermediate (S)-reticuline (Figure 1). Dimeric bisbenzylisoquinoline alkaloids such as berbamunine, which result from the regio- and stereo-selective oxidative carbon–oxygen phenol coupling of N-methylcoclaurine by Cyp80A1, are an exception (Kraus and Kutchan, 1995). The recent isolation of a cDNA from opium poppy encoding a 7-O-methyltransferase specific for norreticuline (N7OMT), but not N-methylated analogs, suggests that precursors for (S)-reticuline might also enter the pathway to form the N-methylated BIA papaverine (J. Ziegler, University of Calgary, personal communication). A cDNA for an O-methyltransferase acting on (S)-reticuline and catalyzing the formation of laudanine was previously isolated from opium poppy (Ounaroon et al., 2003). Interestingly, (R,S)-reticuline 7-O-methyltransferase (7OMT) does not accept N-demethylated BIAs, but is active towards phenolic compounds.

Genes for the biosynthesis of protoberberine alkaloids

Several branch pathways leading to many BIA structural types begin with the formation of (S)-scoulerine by the berberine bridge enzyme (BBE) (Figure 1). BBE cDNAs have been isolated from several sources (Dittrich and Kutchan, 1991; Facchini et al., 1996; Samanani et al., 2005), and the enzyme has been thoroughly characterized (Winkler et al., 2006, 2007). BBE belongs to a novel family of flavoproteins with two covalent binding sites – one histidine and one cysteine – for flavin adenine dinucleotide (FAD). The mid-point redox potential of BBE is higher than that of other flavoproteins due to cysteinylation of the co-factor, and results in hydride abstraction of (S)-reticuline, the first half-reaction toward the conversion to (S)-scoulerine. The biosynthesis of benzophenanthridine alkaloids, such as sanguinarine, is initiated by the formation of two methylenedioxy bridges, resulting in (S)-stylopine (Figure 1). Both reactions are catalyzed by P450-dependent mono-oxygenases, and cDNAs encoding stylopine synthase (Cyp719A2 and Cyp719A3) have been isolated from Eschscholzia californica (Ikezawa et al., 2007). Both recombinant proteins showed the same region-specificity for methylenedioxy bridge formation, but Cyp719A2 only converts (S)-cheilanthifoline to (S)-stylopine, whereas Cyp719A3 also accepts compounds without an existing methylenedioxy bridge. (S)-Stylopine is then converted to (S)-cis-N-methylstylopine by tetrahydroprotoberberine N-methyltransferase (TNMT). A cDNA encoding TNMT was isolated and functionally characterized from opium poppy based on homology to CNMT (Liscombe and Facchini, 2007). TNMT is one of only a few plant enzymes that are able to catalyze the formation of quaternary ammonium compounds, and converts only protoberberine alkaloids with dimethoxy or methylenedioxy functional groups at positions 2/3 and 9/10. Two subsequent P450-dependent hydroxylations yield dihydrosanguinarine, which is oxidized to sanguinarine. However, cDNAs for these final three enzymes have not yet been isolated.

Alternatively, (S)-scoulerine can be methylated to (S)-tetrahydrocolumbamine by scoulerine 9-O-methyltransferase (SOMT) (Figure 1) (Takeshita et al., 1995). Tetrahydrocolumbamine is converted to columbamine, which is methylated by the protoberberine-specific enzyme columbamine O-methyltransferase (CoOMT) to yield palmatine (Morishige et al., 2002). Canadine synthase (Cyp719A1) catalyzes the formation of a methylenedioxy bridge to form canadine from tetrahydrocolumbamine. The inability of Cyp719A1 to accept columbamine as a substrate precludes the involvement of this compound in berberine biosynthesis. (S)-Canadine is ultimately oxidized to berberine.

Genes for the biosynthesis of morphinan alkaloids

Conversion of (S)-reticuline to its (R) epimer is required to initiate the morphinan alkaloid biosynthetic pathway (Figure 1). The epimerization of (S)-reticuline is a two-step process, involving the oxidation of (S)-reticuline by 1,2-dehydroreticuline synthase, and the reduction of 1,2-dehydroreticuline to (R)-reticuline (De-Eknamkul and Zenk, 1992; Hirata et al., 2004). However, cDNAs for these enzymes have not yet been identified. Intramolecular carbon–carbon phenol coupling between the C2 of the benzyl moiety and the C4 of the isoquinoline moiety leads to salutaridine (Gerardy and Zenk, 1993a). A cDNA encoding salutaridine synthase (Cyp719B1) was recently isolated from opium poppy (J. Ziegler, University of Calgary, personal communication). The protein shows high homology to the methylenedioxybridge-forming P450-dependent enzymes. Salutaridine reductase (SalR) converts salutaridine to salutaridinol, and study of a cDNA for SalR isolated from opium poppy has shown that the enzyme belongs to the family of short-chain dehydrogenases/reductases (SDR). The next step in the pathway is catalyzed by salutaridinol 7-O-acetyltransferase (SalAT), which specifically acetylates the 7(S) epimer of salutaridinol to salutaridinol-7-O-acetate. The SalAT cDNA shows considerable homology to the BAHD family of acetyltransferases (Grothe et al., 2001). Depending on pH, the introduced acetyl group can be spontaneously eliminated, leading to the formation of an oxide bridge between C4 and C5 to yield thebaine, the first pentacyclic morphinan alkaloid. The final steps in morphine biosynthesis consist of two demethylations and one reduction. The demethylations are not yet understood and no enzymes capable of catalyzing either reaction have been detected. However, cDNAs for codeinone reductase (COR), which belongs to the aldo-keto reductase family, have been identified in opium poppy (Unterlinner et al., 1999).

Monoterpenoid indole alkaloid metabolism

  1. Top of page
  2. Summary
  3. Introduction
  4. Benzylisoquinoline alkaloid metabolism
  5. Monoterpenoid indole alkaloid metabolism
  6. The role of ‘omics’ technologies to study plant alkaloid biosynthesis
  7. Compartmentalization and trafficking
  8. Gene regulation and signal transduction
  9. Metabolic engineering of MIA and BIA biosynthesis
  10. Outlook
  11. Acknowledgements
  12. References

Genes for the MEP and mevalonic acid pathways

Monoterpenoid indole alkaloids are condensation products of a nitrogen-containing indole moiety derived from tryptamine and a monoterpenoid component derived from the iridoid glucoside secologanin (Figure 2). Incorporation experiments with isotopically labelled glucose in C. roseus cell cultures showed that the initial steps for biosynthesis of the monoterpenoid backbone proceed via the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway (Contin et al., 1998). Four separate cDNAs involved in the MEP pathway – encoding deoxyxylulose 5-phosphate synthase (DXS) (Chahed et al., 2000), deoxyxylulose 5-phosphate reductoisomerase (DXR) (Veau et al., 2000), methylerythritol 2,4-diphosphate synthase (MECS) (Veau et al., 2000) and hydroxymethylbutenyl diphosphate synthase (HDS) (Oudin et al., 2007b) – have been isolated. However, a cDNA for only one mevalonic acid (MVA) pathway enzyme, 3-hydroxy-3-methyl-glutaryl (HMG) CoA reductase (Maldonado-Mendoza et al., 1992), has been reported. Recent sequencing efforts in cDNA libraries derived from leaf epidermis-enriched mRNA have identified 4-diphosphocytidyl-2-C-methyl-d-erythritol synthase (CMS), four MVA pathway genes [3-hydroxy-3-methylglutaryl CoA reductase (HMGR), 3-ketoacyl CoA thiolase (AACT1), acetoacetyl CoA thiolase and HMG CoA synthase (HMGS)] and three genes common to the MEP/MVA pathways [isopentenyl pyrophosphate isomerase (IPPI), farnesyl pyrophosphate synthase (GPPS) and geranyl pyrophosphate synthase (GPS)] (Murata et al., 2008).

image

Figure 2.  Biosynthesis of MIAs including vinblastine, hörhammericine and ajmaline. Enzymes for which corresponding cDNAs have been isolated are shown in red. The key branch-point intermediate dehydrogeissoschizine is highlighted in yellow. Abbreviations: 16OMT, 16-hydroxytabersonine-16-O-methyltransferase; AAE, acetylajmalan esterase; ANAMT, acetylnorajmalan methyltransferase; Cyp71D12, tabersonine 16-hydroxylase; Cyp72A1, secologanin synthase; Cyp76B6, geraniol 10-hydroxylase; D4H, desacetoxyvindoline 4-hydroxylase; DAT, deacetylvindoline 4-O-acetyltransferase; DHVR, dihydrovomilenine reductase; LAMT, loganic acid methyltransferase; MAT, minovincinine acetyltransferase; NAMT, norajmalan methyltransferase; NMT, N-methyltransferase; PER, peroxidase; PNAE, polyneuridine aldehyde esterase; SBE, sarpagan bridge enzyme; SGD, strictosidine β-d-glucosidase; STR, strictosidine synthase; TDC, tryptophan decarboxylase; VH, vinorine hydroxylase; VR, vomilenine reductase; VS, vinorine synthase. Although not shown in the figure, NADPH cytochrome c reductase (CPR) is involved in the Cyp-catalyzed reactions.

Download figure to PowerPoint

Genes for the biosynthesis of secologanin

Four cDNAs involved in secologanin biosynthesis and encoding P450-dependent geraniol 10-hydroxylase (Cyp76B6) (Collu et al., 2001), acyclic monoterpene primary alcohol dehydrogenase (ADH) (Ikeda et al., 1991), loganic acid methyltransferase (LAMT) (Murata et al., 2008) and P450-dependent secologanin synthase (Cyp72A1) (Irmler et al., 2000) have been isolated. Of particular interest is the recent substrate specificity and kinetic analysis of LAMT (Murata et al., 2007), which identified loganic acid as the preferred iridoid substrate in C. roseus, suggesting that O-methylation occurs after (Madyastha et al., 1973) rather than before (Yamamoto et al., 1998) hydroxylation of 7-deoxyloganic acid in this species (Figure 2). Candidate cDNAs encoding 10-oxogeranial cyclase, and several P450 mono-oxygenases potentially involved in the conversion of iridodial to loganic acid, were also identified (Murata et al., 2007).

Monoterpenoid indole alkaloid biosynthesis in C. roseus

Strictosidine, the common precursor to all MIAs, is formed by a Pictet–Spengler condensation of tryptamine and secologanin (Figure 2). Tryptophan decarboxylase (TDC) (De Luca et al., 1989) converts tryptophan to tryptamine, which is then coupled to secologanin by strictosidine synthase (STR) (Kutchan et al., 1988; McKnight et al., 1990) to form strictosidine, the central intermediate leading to all MIAs (Ma et al., 2006). C. roseus accumulates at least 130 MIAs, which are detected in small quantities in various organs and in specialized cell cultures (van der Heijden et al., 2004). Strictosidine β-glucosidase (SGD) is a remarkable strictosidine-specific enzyme that yields a highly reactive ring-opened dialdehyde intermediate that is involved in the biosynthesis of MIAs with corynantheine, iboga and aspidosperma chemical backbones (Geerlings et al., 2000). However, little is known about the mechanisms or enzymes involved in directing the formation of each type. The branch pathways leading from the branch-point intermediate dehydrogeissoschizine to tabersonine and catharanthine remain uncharacterized.

In contrast, the biochemistry of the six-step enzymatic conversion of tabersonine to vindoline has been studied extensively (Loyola-Vargas et al., 2007). The sequence of reactions involves the ordered conversion of tabersonine to 16-hydroxytabersonine by cytochrome P450-dependent tabersonine-16-hydroxylase (Cyp71D12) (Schröder et al., 1999), followed by 16-hydroxytabersonine-16-O-methyltransferase (16OMT) (Levac et al., 2008). The substrate specificity and kinetic properties of 16OMT suggest it has a high affinity for both 16-hydroxytabersonine and SAM. As 16OMT does not accept 16-hydroxy-2,3-dihydrotabersonine as a substrate, O-methylation might occur prior to substitution of the 2,3 position on the molecule (Levac et al., 2008). The conversion of 16-methoxytabersonine to 16-methoxy-2,3-dihydrotabersonine occurs via an uncharacterized oxidation. The subsequent step involves a chloroplast thylakoid membrane-associated N-methyltransferase to yield desacetoxyvindoline. The two terminal reactions in vindoline biosynthesis involve a light-regulated desacetoxyvindoline 4-hydroxylase (D4H) (Vazquez-Flota et al., 1997) and deacetylvindoline 4-O-acetyltransferase (DAT) (St-Pierre et al., 1998). Detection of 16-demethoxyvindoline in C. roseus suggests that the last four reactions in vindoline biosynthesis can transform tabersonine as well as 16-methoxytabersonine into their respective end products.

In contrast to C. roseus leaves and stems, which produce vindoline, underground tissues accumulate other tabersonine derivatives including lochnericine and hörhammericine (Figure 2). An early step in this pathway involves tabersonine 6,7-epoxidase, which catalyzes the NADPH- and molecular oxygen-dependent conversion of tabersonine to lochnericine. This P450 mono-oxygenase was associated with microsomes of transformed C. roseus hairy root cultures (Rodriguez et al., 2003). A cDNA for the root tip-specific minovincinine-O-acetyltransferase (MAT) involved in the biosynthesis of 6,7-dehydroechitovenine and/or 19-O-acetylhörhammericine has been functionally characterized (Laflamme et al., 2001).

Monoterpenoid indole alkaloid biosynthesis in Rauvolfia serpentina

The biosynthesis of ajmaline in cell cultures of Rauvolfia serpentina involves ten well-characterized enzymatic steps (Figure 2) (Ruppert et al., 2005). In addition, cDNAs have been isolated for six genes involved in general MIA and ajmaline metabolism in R. serpentina– STR, SGD, polyneuridine aldehyde esterase (PNAE), vinorine synthase (VS), cytochrome P450 reductase (CPR) and acetylajmalan acetylesterase (AAE) (Ruppert et al., 2005). The three-dimensional structures for several of these enzymes have been elucidated, which has provided insights into the reaction mechanisms involved in MIA biosynthesis (Stöckigt et al., 2007).

The role of ‘omics’ technologies to study plant alkaloid biosynthesis

  1. Top of page
  2. Summary
  3. Introduction
  4. Benzylisoquinoline alkaloid metabolism
  5. Monoterpenoid indole alkaloid metabolism
  6. The role of ‘omics’ technologies to study plant alkaloid biosynthesis
  7. Compartmentalization and trafficking
  8. Gene regulation and signal transduction
  9. Metabolic engineering of MIA and BIA biosynthesis
  10. Outlook
  11. Acknowledgements
  12. References

The Arabidopsis (Arabidopsis thaliana) genome sequence has been available since 2000, and projects targeting other model species including rice, corn, soybean, poplar and the legume Medicago truncatula are at various stages of completion. However, genome sequence databases are not yet available for plants that produce complex alkaloids. Nevertheless, application of high-throughput technologies such as genome sequencing, expressed sequence tag (EST) and DNA microarray analyses, proteomics and metabolomics has recently accelerated the discovery of new genes and biological processes involved in plant secondary metabolism.

Expressed sequence tags

Compared with model plants such as Arabidopsis, rice and tomato, the number of ESTs for alkaloid-producing plants is relatively low. An exception is tobacco, for which more than one million ESTs are publicly available due to the importance of this plant as a model for investigation of plant–pathogen and plant–virus interactions. As alkaloid metabolism is so diverse and few research groups work on the same plant species, large-scale EST projects are limited. However, extensive EST databases are now available for opium poppy (and several related BIA-producing plants) and for C. roseus. The release of a database consisting of 4500 ESTs derived from the laticifers of morphine-accumulating opium poppy was reported in 2001 (Pilatzke-Wunderlich and Nessler, 2001). Further efforts with this plant have yielded a total of 25 000 ESTs from seedlings, stems, roots and elicitor-treated cell cultures (Ziegler et al., 2005, 2006; Zulak et al., 2007). A recent floral genome project contributed 11 000 ESTs from Eschscholzia californica (Carlson et al., 2006), and the release of a Coptis japonica database has also been reported (Inui et al., 2007; Kato et al., 2007). NAPGEN (Natural Products Genomic Research), a consortium of Canadian investigators, has recently established a database of over 400 000 ESTs from a wide variety of plants that accumulate potentially health-related bioactive metabolites. Within this project, more than 46 000 ESTs were reported from cell cultures for eight BIA-accumulating species of four plant families.

Over 25 000 C. roseus ESTs have been produced by partial sequencing of cDNA libraries prepared from whole organs and tissues (Murata et al., 2006; Rischer et al., 2006; Shukla et al., 2006). Previously, only 372 C. roseus sequences were available in public databases. Despite the high MIA biosynthetic activity of the selected C. roseus tissues (Laflamme et al., 2001; St-Pierre et al., 1999), no known transcripts involved in MIA biosynthesis, except for those encoding Cyp76B6 and SGD, were represented in the clones obtained from young leaf, root tip and other cDNA libraries (Murata et al., 2006; Rischer et al., 2006; Shukla et al., 2006). The absence of MIA biosynthesis genes is consistent with results obtained in cDNA/amplified fragment length polymorphism studies conducted with induced C. roseus cell cultures (Rischer et al., 2006). Together, these results suggest that gene transcripts relevant to MIA metabolism are rare in induced cell cultures or in young leaves/root tips compared with transcripts encoding cellular components that are not related to MIA biosynthesis. The isolation of leaf epidermis mRNA by carborundum abrasion facilitated the production of a representative cDNA library, and more than 10 000 unique ESTs were identified (Murata et al., 2008 and unpublished results). Remarkably the leaf epidermis-enriched cDNA library contained virtually all known MIA biosynthesis and regulatory genes (Murata et al., 2008).

Transcriptome analysis

The earliest attempt to identify preferentially expressed genes in alkaloid-producing plants involved differential screening by subtractive hybridization of low and normal alkaloid-producing lines of tobacco to isolate and characterize putrescine N-methyltransferase, an early step in nicotine biosynthesis, (Hibi et al., 1994). Several other developments first pioneered in tobacco (Goossens and Rischer, 2007) are now being applied to opium poppy, C. roseus and other medicinal plants. Recently, a cDNA/amplified fragment length polymorphism method was used to profile cell cultures in terms of metabolites and transcripts at various times after jasmonate (JA) treatment (Rischer et al., 2006). The analysis for MIA pathway genes included the identification of a number of MEP pathway genes (DXS, HDS, MECS), as well as Cyp76B6, which commit geraniol to the secoiridoid pathway, and SGD, which converts strictosidine to its aglycone. Other putative MIA pathway genes were identified, but require functional characterization. The metabolite and transcript profiles were integrated, which yielded some intriguing correlations, but the results are equivocal without more accurate tools to select candidate MIA biosynthetic or regulatory genes for further study. More recent efforts included small-scale transcriptome analyses using cDNA libraries produced from C. roseus mRNA extracted from leaves and roots, respectively (Shukla et al., 2006). Random small-scale sequencing of leaf, root and subtracted cDNAs did not reveal new information about MIA biosynthesis.

DNA macroarray analysis of differential gene expression among morphine-containing opium poppy plants and eight other Papaver species that do not accumulate morphine produced a set of cDNAs with higher expression in opium poppy, one of which encoded 4′OMT (Ziegler et al., 2005) and another encoded SalR (Ziegler et al., 2006). DNA microarray analysis of the top1 mutant variety of opium poppy, which accumulates thebaine and oripavine rather than codeine and morphine, revealed ten down-regulated transcripts, although none appeared to encode proteins responsible for the high-thebaine, low-morphine phenotype (Millgate et al., 2004). Large-scale transcriptome and metabolite profiling has been performed in opium poppy cell cultures treated with a fungal elicitor (Zulak et al., 2007), and in C. roseus and tobacco cell cultures treated with methyl JA (Goossens et al., 2003; Rischer et al., 2006). In all cases, a coordinated increase was observed in the expression of genes implicated in alkaloid metabolism. Moreover, profound changes in the level of gene transcripts encoding primary metabolic enzymes also occurred. Interestingly, transcripts involved in SAM recycling increased in all three systems, indicating a high demand for this co-factor in the modification of alkaloid backbone structures.

Proteomics and metabolomics

Proteomics has been used to identify proteins found in the exuded latex of opium poppy (Decker et al., 2000), and subsequently to characterize 6OMT and 7OMT within the protein population (Ounaroon et al., 2003). A proteomics approach was also undertaken to identify novel proteins involved in alkaloid biosynthesis during the growth cycle of C. roseus cell cultures (Jacobs et al., 2005). Eighty-eight protein spots were selected for identification by MALDI-MS/MS, of which 58 were identified, including two STR isoforms and tryptophan synthase. These studies demonstrate that proteomics has the potential to become a valuable approach once protein databases are established.

Several pioneering metabolomic analyses have recently been performed on MIA-producing plant species, including C. roseus (Choi et al., 2004) and Strychnos spp. (Frederich et al., 2004). Comprehensive metabolic profiling of various phytoplasma-treated C. roseus leaves by NMR spectroscopy followed by principle component analysis identified compounds present at different levels in infected versus healthy tissues (Choi et al., 2004). Phytoplasma infection caused an increase in phenylpropanoid and MIA pathway intermediates and products, including chlorogenic acid, loganic acid, secologanin and vindoline. Another metabolomic study was conducted using liquid chromatography coupled with photodiode array detection and ESI-MS on methanol extracts from Ophiorrhiza pumila, Nothapodytes foetida and Camptotheca acuminata to compare the profiles of camptothecin-related alkaloids being produced (Yamazaki et al., 2003). Considerable differences were detected in the constituents of the various plants, hairy roots and cell cultures.

Compartmentalization and trafficking

  1. Top of page
  2. Summary
  3. Introduction
  4. Benzylisoquinoline alkaloid metabolism
  5. Monoterpenoid indole alkaloid metabolism
  6. The role of ‘omics’ technologies to study plant alkaloid biosynthesis
  7. Compartmentalization and trafficking
  8. Gene regulation and signal transduction
  9. Metabolic engineering of MIA and BIA biosynthesis
  10. Outlook
  11. Acknowledgements
  12. References

Although the biosynthetic pathways leading to BIAs, MIAs and other alkaloid types are of polyphyletic origin, some intriguing patterns are apparent, including the involvement of multiple cell types for alkaloid biosynthesis and/or accumulation, the targeting of pathway enzymes to multiple subcellular compartments, and the possible ubiquity of multi-enzyme complexes. Such complex organization implies extensive intra- and inter-cellular transport of pathway intermediates, products and even biosynthetic enzymes. Alkaloids generally accumulate in specific cell types due to their cytotoxicity and probable role in plant defense responses. For example, alkaloids are sequestered to isolated idioblasts and laticifers in C. roseus (St-Pierre et al., 1999), root endodermis and stem cortex/pith in Thalictrum flavum (Samanani et al., 2005), and laticifers in opium poppy (Bird et al., 2003). The cellular localization of alkaloid biosynthesis is remarkably diverse and complex. Work on the cellular and developmental complexities and organization of alkaloid biosynthesis in opium poppy and C. roseus, in particular, has established new paradigms in the cell biology of secondary metabolism.

A tale of three cell types in opium poppy

In all organs of opium poppy, BIA accumulation occurs in the articulated laticifers found adjacent or proximal to sieve elements of the phloem (Figure 3) (Facchini and De Luca, 1995). The cytoplasm of laticifers (latex) contains a full complement of cellular organelles and many large vesicles to which alkaloids are sequestered. Although laticifers were long considered the site of BIA biosynthesis and accumulation, the cellular localization of BIA biosynthetic enzymes and gene transcripts has shown that alkaloid synthesis involves other cell types (Bird et al., 2003; Facchini and De Luca, 1995; Weid et al., 2004). Initial in situ hybridization experiments demonstrated localization of TYDC gene transcripts to the phloem, but not to laticifers (Facchini and De Luca, 1995). The morphinan pathway enzymes Cyp719B1 and SalR were also not detected in isolated latex (Gerardy and Zenk, 1993a,b). Seven other biosynthetic enzymes (6OMT, CNMT, CYP80B1, 4′OMT, BBE, SalAT and COR) have been localized to sieve elements in opium poppy and their corresponding gene transcripts to associated companion cells (Figure 3) (Bird et al., 2003; Samanani et al., 2006). The localization of BIA metabolism to phloem cells distinct from laticifers indicates inter-cellular transport of pathway intermediates or products, and is contrary to a long-standing view in plant biology (Figure 4). Previously, sieve elements were not known to support complex metabolism, and were assumed to possess only a limited number of proteins required for cell maintenance and solute transport. The expanding physiological roles for sieve elements include the transport of information macromolecules such as RNA (Jorgensen et al., 1998), and the biosynthesis of jasmonic acid (Hause et al., 2003), ascorbic acid (Hancock et al., 2003) and defense-related compounds (Walz et al., 2004). Sieve elements clearly possess a previously unrealized biochemical potential.

image

Figure 3.  Three-dimensional representation of vascular tissues in opium poppy showing the location of sieve elements (yellow), companion cells and laticifers involved in the biosynthesis and accumulation of BIAs.

Download figure to PowerPoint

image

Figure 4.  BIA biosynthesis involves three cell types in the phloem of opium poppy. (a) Laticifers are adjacent to sieve elements and companion cells. Stem tissue was fixed using potassium permanganate and sectioned for viewing by transmission electron microscopy. Abbreviations: cc, companion cell; cw, cell wall; er, endoplasmic reticulum; la, laticifer; m, mitochondrion; p, plastid; se, sieve element; ser, sieve element reticulum; v, vacuole. Bar = 10 μm. (b) Plasmodesmata create symplastic connections between sieve elements and laticifers. Stem cross-sections were exposed to a mouse β-1,3-glucan monoclonal antibody (green, asterisks) to show plasmodesmata, and a rabbit major latex protein (MLP) polyclonal antibody (red) to identify laticifers. An obliquely oriented sieve plate (green, white arrowhead) identifies the sieve element. Bar = 10 μm.

Download figure to PowerPoint

Thalictrum flavum accumulates the anti-microbial agent berberine and other BIAs. In situ RNA hybridization analysis revealed cell type-specific expression of BIA biosynthetic genes in the roots and rhizomes of T. flavum (Samanani et al., 2005). In roots, gene transcripts for all nine enzymes were localized to the pericycle (the innermost layer of the cortex) and adjacent cortical cells. In rhizomes, all biosynthetic gene transcripts were restricted to the protoderm of leaf primordia. As in opium poppy, biosynthetic gene transcripts in T. flavum were localized to cell types that are different from those in which alkaloids accumulate. In roots, protoberberine alkaloids were restricted to endodermal cells upon initiation of secondary growth, and distributed throughout the pith and cortex in rhizomes. Thus, the cell type-specific localization of BIA biosynthesis and accumulation are temporally and spatially separated in T. flavum roots and rhizomes, respectively. Despite the close phylogenetic relationships between corresponding biosynthetic enzymes (Liscombe et al., 2005), different cell types are involved in BIA synthesis and storage in opium poppy, T. flavum and perhaps Coptis japonica.

The cellular source of isopentenyl diphosphate for secologanin biosynthesis

Two separate routes for IPP biosynthesis have been established in plants – the cytosolic MVA pathway and the plastid-localized MEP pathway (Rohmer, 1999). Strong evidence based on feeding studies with [1-13C]-glucose supports the role of the MEP pathway in supply of IPP precursors for secologanin biosynthesis in C. roseus cell cultures (Contin et al., 1998) and Ophiorrhiza pumila hairy roots (Yamazaki et al., 2004). Other studies conducted with R. serpentina cell cultures suggest that the MVA pathway might contribute up to 5% of the precursors for loganin biosynthesis, which implies that some cross-talk occurs between the two pathways. In situ RNA hybridization studies in leaf showed that gene transcripts encoding four MEP pathway enzymes and geraniol 10-hydroxylase (Cyp76B6) were primarily localized in the internal phloem-associated parenchyma (IPAP) (Burlat et al., 2004; Mahroug et al., 2006; Oudin et al., 2007b). This discovery led to the hypothesis that IPAP cells participate in MIA biosynthesis by supplying an isoprenoid pathway intermediate that is translocated to the leaf epidermis for conversion to secologanin and ultimately MIAs (Figure 5). Identification of this intermediate is essential to substantiate this hypothesis, but this will be difficult without whole-plant genetic engineering capabilities.

image

Figure 5.  Three-dimensional representation of leaf tissues in Catharanthus roseus showing the location of epidermis, idioblasts, laticifers and internal phloem parenchyma (yellow) involved in the biosynthesis and accumulation of MIAs.

Download figure to PowerPoint

MIA biosynthesis involves multiple cell types in above-ground parts of C. roseus

Initial studies based on in situ RNA hybridization and immunocytochemistry established that MIA biosynthesis is developmentally regulated and compartmentalized in more than one leaf or stem cell type in C. roseus (St-Pierre et al., 1999). TDC and STR transcripts were localized to the epidermis of leaves, stems and flower buds, whereas D4H and DAT transcripts were associated with specialized laticifer and idioblast cells in these organs (Figure 5). In underground tissues, transcripts encoding TDC and STR1 (St-Pierre et al., 1999) and MAT (Laflamme et al., 2001) were localized to the protoderm and cortical cells around the root apical meristem. Neither D4H nor DAT transcripts nor gene products were detected in roots (St-Pierre et al., 1999), which is consistent with the accumulation of vindoline in aerial organs only. The involvement of leaf/stem epidermis and idioblasts/laticifers suggests inter-cellular translocation of at least one pathway intermediate in MIA metabolism (Figures 5 and 6).

image

Figure 6.  Putative localization and inter-cellular trafficking of MIA and other metabolic pathways. Catharanthus roseus leaf epidermal cells express alkaloid, MVA, triterpene, very-long-chain fatty acids (VLFA) and flavonoid biosynthetic pathways. Triterpenes and VFLAs are secreted to the waxy cuticle, whereas flavonoids accumulate in epidermal cell vacuoles. The model proposes that most of the vindoline pathway up to and including 16-methoxytabersonine is expressed in the leaf epidermis, and an undetermined intermediate is then transported to adjacent mesophyll cells, specialized idioblasts and/or laticifers, for final elaboration into vindoline. The internal phloem-associated parenchyma (IPAP) preferentially expresses the MEP pathway and Cyp76B6, which raises questions about the roles played by the epidermis and IPAP in supplying IPP for secologanin biosynthesis.

Download figure to PowerPoint

Recently, laser-capture microdissection (LCM) was used to isolate individual cell types from C. roseus leaves and examine the role of each in relation to MIA biosynthesis (Murata and De Luca, 2005). LCM produced sufficient and relatively homogeneous epidermis, idioblasts, laticifers, mesophyll and vascular tissues to allow the isolation of RNA and subsequent synthesis of cell-specific cDNAs. TDC, STR, D4H and DAT transcript levels in these cDNA populations were consistent with those determined by in situ RNA hybridization (St-Pierre et al., 1999). Preferential expression of Cyp71D12 and SGD and the detection of Cyp76B6 in leaf epidermis suggested that the entire pathway from geraniol to 16-hydroxytabersonine might be completed in this cell type (Murata and De Luca, 2005). However, the low levels of expression of Cyp76B6 in leaf epidermis compared with IPAP, together with preferential expression of the MEP pathway in these cells (Burlat et al., 2004; Mahroug et al., 2006; Oudin et al., 2007b), has raised questions about the supply of iridoid precursors for secologanin biosynthesis (Figure 6).

Carborundum abrasion has been used as a novel and complementary approach to obtain epidermis-enriched leaf extracts to measure alkaloid metabolite, enzyme activity and gene expression levels (Murata and De Luca, 2005). Use of this technique showed that levels of tabersonine and 16-methoxytabersonine were higher in leaf epidermis-enriched extracts compared with vindoline and catharanthine. Similarly, 16OMT was found predominantly in C. roseus leaf epidermis, in contrast to DAT and the N-methyltransferase involved in the vindoline pathway, which were only found in whole-leaf extracts (Murata and De Luca, 2005).

MIA pathways in leaf epidermis and cortical cells of the root apical meristem

Catharanthus roseus hairy roots accumulate tabersonine, lochnericine, hörhammericine and catharanthine, demonstrating the de novo biosynthetic potential of roots to produce these alkaloids (Magnotta et al., 2007; Morgan and Shanks, 1999). However, it has not been determined whether leaf epidermis is also involved in the biosynthesis of catharanthine, or whether this alkaloid is synthesized in roots and is then transported to leaves. However, the biosynthetic competence of C. roseus shoot cultures to produce both catharanthine and vindoline (Hirata et al., 1990) has been reported, suggesting that the leaf epidermis might support the formation of both alkaloids.

Carborundum abrasion to study the C. roseus leaf epidermone

Several approaches have been used to clone 16OMT, which catalyzes the second step in the conversion of tabersonine to vindoline. Homology-based methods (Cacacea et al., 2003; Schröder et al., 2004) and attempts to purify the protein from cultured cells were unsuccessful (Schröder et al., 2004). Use of caborundum abrasion for large-scale isolation of leaf epidermal proteins facilitated the purification of 16OMT and isolation of the cognate cDNA (Levac et al., 2008). Recombinant 16OMT shows a narrow substrate range and high affinity for 16-hydroxytabersonine. The caborundum abrasion approach also clearly demonstrates that 16OMT is predominantly expressed in C. roseus leaf epidermis. The biochemical specialization of young leaf epidermis for MIA biosynthesis (Levac et al., 2008) suggests that the remaining uncharacterized biosynthetic and regulatory genes in the MIA pathway will be found in the epidermis-enriched EST database. A homology-based cloning strategy was used to identify LAMT, the second-to-last step in secologanin biosynthesis, in the EST database (Murata et al., 2008). As expected, LAMT activity was enriched in protein extracts from leaf epidermis compared with whole-leaf extracts.

In addition to MIA biosynthesis, several MVA pathway genes and genes common to downstream pathways were identified in the EST database (Murata et al., 2008). Enrichment of the cytosolic isoprenoid and related pathways appears important for leaf epidermis-localized biosynthesis of the oleanane-type triterpenes, which are secreted into to the cuticular wax layer of C. roseus leaves (Figure 6). These triterpene alcohols are composed primarily of ursolic acid and small amounts of oleanolic acid (Usia et al., 2005), and constitute approximately 2.5% (dry weight) of the young C. roseus leaves (Murata et al., 2008). In support of this hypothesis, ESTs representing a putative squalene mono-oxygenase and β-amyrin synthase, which are required for oleanane-type triterpene biosynthesis, were identified in the leaf epidermis-enriched cDNA library (Murata et al., 2008). The specialization of leaf epidermis for triterpenoid biosynthesis and accumulation raises important issues and questions regarding the ability of the epidermis to accommodate the MEP pathway for secologanin biosynthesis. Extensive sequencing of leaf epidermis enriched C. roseus cDNA libraries showed that several steps in the MVA pathway were well-represented, whereas only a single MEP pathway step and a single G10H EST could be identified (Murata et al., 2008). These results add support to in situ hybridization studies that show preferential expression of the MEP pathway and G10H in IPAP cells (Burlat et al., 2004). However, this remains to be proven by more rigorous experimentation to establish whether the pathways for triterpenoid and the entire pathway for secoiridoid biosynthesis co-exist in the same cell type (Murata et al., 2006, 2008) or whether part of the secoiridoid pathway occurs in IPAP cells (Mahroug et al., 2007).

The biochemical capacity of C. roseus leaf epidermis for flavonoid biosynthesis was first suggested by localization of transcripts encoding phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H) and chalcone synthase (CHS) in epidermal cells (Figure 6) (Mahroug et al., 2006). Nine putative genes encoding PAL, C4H, CHS, chalcone reductase, flavanone 3-hydroxylase, 2-hydroxyisoflavanone dehydratase, 2′-hydroxyisoflavone/dihydroflavonol reductase, quercetin 3-O-glucoside-6′-O-malonyltransferase and anthocyanidin rhamnosyl transferase were also identified in the leaf epidermis-enriched EST database (Murata et al., 2008). ESTs encoding more than 20 enzymes involved in the pathway for very-long-chain fatty acid biosynthesis were also detected in the leaf epidermis-enriched cDNA library (Figure 6) (Murata et al., 2008). These results clearly illustrate the biochemical potential of C. roseus leaf epidermis for the production of diverse secondary metabolites.

Subcellular compartmentalization of alkaloid biosynthetic enzymes

Several enzymes involved in BIA biosynthesis are associated with a subcellular compartment other than the cytosol. For the sanguinarine pathway, density gradient fractionation suggested the localization of BBE and P450 mono-oxygenases to microsomes with a density slightly higher than that of typical endoplasmic reticulum (ER) (Amann et al., 1986; Bauer and Zenk, 1991; Rueffer and Zenk, 1987; Tanahashi and Zenk, 1990). Although BBE is not an integral membrane protein, it is initially targeted to the ER and subsequently sorted to a vacuolar compartment (Bird and Facchini, 2001). NCS has also been predicted to possess a signal peptide targeting the ER (Samanani et al., 2004), and the final oxidation of alkaloids, such as dihydrosanguinarine, probably occurs in an ER-derived compartment (Amann et al., 1986). The association of BIA biosynthetic enzymes with endomembranes led to speculation that specialized alkaloid-synthesizing vesicles are present in alkaloid-producing cells (Amann et al., 1986). Recently, however, Cyp80B3, BBE and sanguinarine were co-localized with the ER in opium poppy cell cultures (Alcantara et al., 2005). As the pH optimum for BBE is approximately 8.8 (Steffens et al., 1985), sanguinarine biosynthesis is unlikely to involve the vacuole; thus, BIA metabolism could be entirely associated with the ER. Indeed, the vacuole might not even be the site of BIA accumulation in cultured cells, as previously thought. Instead, sanguinarine and related alkaloids could be secreted and bound to cell-wall components, re-absorbed, reduced to less toxic dihydrosanguinarine, and further metabolized (Weiss et al., 2006).

In addition to various cell types, enzymes involved in MIA biosynthesis in C. roseus have also been localized to at least five subcellular compartments: TDC, D4H and DAT are found in the cytosol (De Luca and Cutler, 1987), STR and the peroxidase involved in the coupling of MIA monomers were localized to the vacuole (McKnight et al., 1991; Sottomayor et al., 1998), SGD is a soluble enzyme that is purported to associate with the cytoplasmic face of the endoplasmic reticulum (Stevens et al., 1993), the P450 mono-oxygenases Cyp76B6, Cyp72A2 and Cyp71D12 are integral endomembrane proteins (Collu et al., 2001; St-Pierre and De Luca, 1995; Yamamoto et al., 2000), and the N-methyltransferase involved in vindoline biosynthesis is localized to chloroplast thylakoid membranes (Dethier and De Luca, 1993).

Enzyme complexes and metabolic channels

It is generally assumed that many metabolic enzymes interact physically or are in close proximity with other enzymes that participate in common pathways (Winkel, 2004). Theoretically, the existence of multi-enzyme complexes – also known as metabolic complexes/channels or metabolons – promote more efficient cellular metabolism. RNAi-mediated silencing of COR genes suggested the possible existence of a multi-enzyme complex in BIA metabolism (Allen et al., 2004). Although seven enzymatic steps occur between (S)-reticuline and codeinone (the substrate for COR), only (S)-reticuline accumulated at elevated levels. No intermediates between (S)-reticuline and morphine were detected. Removal of COR was suggested to disrupt a metabolic channel composed of morphinan branch pathway enzymes, resulting in the accumulation of an alkaloid intermediate produced by enzymes that are not part of the same complex. Interestingly, thebaine and oripavine, which are both intermediates upstream of substrates acted upon by COR, accumulate to high levels in some opium poppy cultivars (Millgate et al., 2004); thus, an accumulation of morphinan branch pathway intermediates was expected. An alternative interpretation not considered by Allen et al. (2004) is the possible suppression of 1,2-dehydroreticuline reductase as a side-effect of silencing COR. 1,2-Dehydroreticuline reductase is one of two enzymes involved in the epimerization of (S)-reticuline to (R)-reticuline. The potential homology between the two reductases could lead to co-silencing.

Gene regulation and signal transduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Benzylisoquinoline alkaloid metabolism
  5. Monoterpenoid indole alkaloid metabolism
  6. The role of ‘omics’ technologies to study plant alkaloid biosynthesis
  7. Compartmentalization and trafficking
  8. Gene regulation and signal transduction
  9. Metabolic engineering of MIA and BIA biosynthesis
  10. Outlook
  11. Acknowledgements
  12. References

The biosynthesis and accumulation of MIAs and BIAs are coordinately regulated by development-, tissue- and environment-specific factors (Memelink and Gantet, 2007). Early studies with opium poppy and C. roseus cell cultures showed that alkaloid biosynthesis and accumulation could be triggered by alterations to the composition of the culture medium or by treatment with specific elicitors such as JA or fungal cell-wall components (El-Sayed and Verpoorte, 2007; Facchini, 2006). Similarly, studies with hairy root cultures, organ cultures, seedlings and plant organs at various stages of development have shown that BIA and MIA metabolism are temporally and developmentally regulated.

Regulation of gene expression in MIA biosynthesis

Conserved regulatory elements in the STR promoter were used to isolate various regulatory genes that control JA- and elicitor-inducible expression of MIA biosynthesis in C. roseus cell cultures (Memelink and Gantet, 2007). A yeast one-hybrid screen using a JA- and elicitor-responsive regulatory element from the STR1 promoter was used to identify a cDNA encoding the ORCA2 (octadecanoid-responsive C. roseus APETALA-domain protein 2) transcription factor (Menke et al., 1999). In another study, T-DNA activation tagging (van der Fits et al., 2001) led to identification of the cDNA encoding ORCA3 (van der Fits and Memelink, 2000), which regulates several early steps in MIA biosynthesis. Over-expression of ORCA3 in transformed C. roseus cell cultures increased the expression of several MIA pathway genes (van der Fits and Memelink, 2000). Although the possible overlapping functions of ORCA3 and ORCA2 must still be determined, an immediate consequence of treating C. roseus cell cultures with JA is a post-translational modification of ORCA3, possibly via phosphorylation (Memelink and Gantet, 2007).

Several other transcription factors appear to participate in the elicitor-mediated activation of MIA biosynthesis in C. roseus cell cultures (Memelink and Gantet, 2007). A CrBPF1 transcription factor similar to the MYB-like factor BPF1 from parsley was shown to bind to an alternative STR1 promoter site that lies upstream of the JA- and elicitor-responsive regulatory element (van der Fits et al., 2000). CrBPF1 appears to enhance elicitor-mediated STR1 gene expression, but cannot replace ORCA3 to activate MIA biosynthesis in cell cultures and proceeds via a JA-independent signaling pathway (Menke et al., 1999). The G-box, a third regulatory element found between the BPF1 and ORCA binding sites in the STR1 promoter, was used in a yeast one-hybrid screen to isolate CrGBS (G-box binding factor) and CrMYC transcription factors (Ouwerkerk and Memelink, 1999). Transient assays suggested that these transcription factors repress STR1 gene expression, although their exact roles in MIA biosynthesis are not fully characterized (Sibéril et al., 2001). Additional yeast one-hybrid screening using another elicitor-responsive element from the TDC promoter identified three Cys2/His2-type zinc finger proteins, which appear to repress both the TDC and STR1 promoters (Pauw et al., 2004).

Transcriptional regulation of BIA biosynthesis

The recent application of transcript profiling based on DNA macroarrays constructed from ESTs has led to isolation of the first transcription factor putatively involved in the regulation of BIA metabolism. Transcripts encoding a subset of transcriptional regulators showed coordinated expression with respect to BIA biosynthetic genes in berberine-producing Coptis japonica cell cultures. One of these, a WRKY transcription factor, was shown by RNAi and over-expression analysis to specifically regulate the expression of BIA biosynthetic genes (Kato et al., 2007).

Signal transduction

A yeast-derived elicitor was shown to activate the protein phosphorylation- and calcium flux-dependent production of reactive oxygen species as a second messenger in C. roseus cell cultures (Pauw et al., 2004). However, generation of reactive oxygen species was not required for the elicitor-mediated induction of alkaloid biosynthetic genes, suggesting that the oxidative burst is coupled to activation of other defense genes. The necessity for a functional octadecanoid pathway was shown in C. roseus cells cultured in an auxin-starved medium (Gantet et al., 1998). Addition of auxin to cell cultures inhibits accumulation of ajmalicine, but stimulates the accumulation other MIAs such as tabersonine and catharanthine (Rischer et al., 2006). However, exogenous addition of MeJA to auxin-treated cells restores their ability to produce alkaloids. Alkaloid production is also reduced in auxin-starved cells treated with octadecanoid pathway inhibitors, suggesting that JAs are produced in response to auxin depletion and function to coordinate biochemical events that lead to alkaloid biosynthesis. A role for the CaaX-prenyltransferases, protein farnesyltransferase (PFT) and type I protein geranylgeranyltransferase (PGGT-I), in regulating early MIA biosynthetic genes, including those encoding enzymes involved of the MEP pathway, has been demonstrated by RNAi-mediated silencing of the corresponding genes in C. roseus cell cultures (Courdavault et al., 2005). Another component of the JA signal transduction cascade also involves post-translational events that activate an uncharacterized upstream transcription factor that up-regulates ORCA3 gene expression (Vom Endt et al., 2002). Recent studies using one-hybrid transcription factor screening with a bipartite JA-responsive element have identified an AT-hook transcription factor that may be part of the upstream components that activate ORCA3 gene expression (Vom Endt et al., 2007).

Considerable effort has been focused on events associated with the induction of BIA metabolism in E. californica cell cultures, which accumulate benzophenanthridine alkaloids such as sanguinarine in response to treatment with a fungal elicitor. Two signal transduction pathways were identified. One cascade is JA-dependent and responds to high elicitor concentrations. The other is JA-independent and is triggered by low elicitor concentrations (Färber et al., 2003). The JA-independent pathway involves Gα proteins that interact and activate phospholipase A2, which leads to a transient efflux of protons from the vacuole and subsequent activation of other cytoplasmic components (Heinze et al., 2007; Viehweger et al., 2006).

Metabolic engineering of MIA and BIA biosynthesis

  1. Top of page
  2. Summary
  3. Introduction
  4. Benzylisoquinoline alkaloid metabolism
  5. Monoterpenoid indole alkaloid metabolism
  6. The role of ‘omics’ technologies to study plant alkaloid biosynthesis
  7. Compartmentalization and trafficking
  8. Gene regulation and signal transduction
  9. Metabolic engineering of MIA and BIA biosynthesis
  10. Outlook
  11. Acknowledgements
  12. References

Metabolic engineering is defined as the improvement of cellular activities by the manipulation of enzymatic, transport or regulatory functions using recombinant DNA technology. Several biosynthetic and regulatory genes have been used to genetically alter the production of alkaloids in opium poppy and C. roseus. Transgenic plants with altered or novel enzyme activities have also been used as a tool to study the metabolic control architecture of alkaloid pathways (Hughes et al., 2004). Procedures have been developed for the transformation of opium poppy plants (Chitty et al., 2003; Facchini et al., 2008; Park and Facchini, 2000a), root cultures (Le Flem-Bonhomme et al., 2004; Park and Facchini, 2000b) and cell cultures (Belny et al., 1997). Stable transformation protocols for C. roseus are presently restricted to roots and cell cultures (Pasquali et al., 2006; Shanks et al., 1998; Zárate and Verpoorte, 2007; Zhao and Verpoorte, 2007). Genetic transformation of opium poppy and C. roseus has provided the opportunity to alter the activity of individual enzymes of BIA biosynthesis, and to examine the consequences of such modifications on the accumulation of pathway products and intermediates.

Metabolic engineering of BIA biosynthesis

Over-expression of BBE in E. californica root cultures resulted in an increased accumulation of downstream alkaloids and decreased levels of certain amino acids (Park et al., 2003). Conversely, antisense suppression of BBE expression led to non-detectable levels of downstream metabolites and increased cellular amino acid pools, revealing the impact of perturbations in alkaloid metabolism on primary metabolism (Park et al., 2002). Interestingly, antisense suppression of BBE did not result in accumulation of its substrate (S)-reticuline or any other upstream alkaloid, suggesting that metabolic channels might be disrupted in the absence of BBE. In contrast, RNAi lines targeting BBE in E. californica cell cultures resulted in elevated levels of (S)-reticuline (Fujii et al., 2007). Furthermore, the detection of laudanine suggested that the pathway was redirected towards the O-methylation of reticuline by 7OMT. Transgenic opium poppy plants expressing an antisense BBE construct showed increased flux into the morphinan and tetrahydrobenzylisoquinoline branch pathways (Frick et al., 2004). Surprisingly, the BBE reaction product (S)-scoulerine also accumulated in the latex of these transgenic plants, although the roots showed no alterations in metabolite profile. Whether these results are due to an additional role for BBE in BIA metabolism remains unclear.

Opium poppy plants transformed with over-expressed or antisense-suppressed Cyp80B3, which encodes N-methylcoclaurine 3′-hydroxylase, also showed substantial modulations in alkaloid content, but not profile (Frick et al., 2007). Over-expression of COR1, the penultimate step in morphine biosynthesis, also resulted in opium poppy plants with increased levels of morphine, codeine, and (for unexplained reasons) thebaine, which is upstream of COR1 in the pathway (Larkin et al., 2007). Large-scale expression data suggest that over-expression of one alkaloid biosynthetic gene might cause coordinated transcriptional induction of other pathway genes. In RNAi-silenced COR1 plants, expression of other known BIA biosynthetic genes from the pathway was unaffected (Allen et al., 2004). However, for unknown reasons, the plants accumulated the far-upstream intermediate (S)-reticuline rather than codeinone, the substrate of COR. The presence of multi-enzyme complexes in morphine biosynthesis is supported by experiments involving the suppression of SalAT. RNAi-silenced SalAT plants showed accumulation of salutaridine, which is normally not abundant in opium poppy plants (Allen et al., 2007). This is surprising as salutaridinol, the substrate for SalAT, did not accumulate (Figure 1). Salutaridine might be channelled to thebaine via an enzyme complex that includes SalR and SalAT.

Constitutive expression of two O-methyltransferases from the early BIA pathway in E. californica cells suggested a rate-limiting role for 6OMT (Inui et al., 2007). Constitutive over-expression of 6OMT led to increased alkaloid content. In contrast, over-expression of 4′OMT had little effect. The E. californica cell culture used in this study appeared to lack a functional 6OMT, which might explain the strong effect of over-expressing a 6OMT transgene. Subsequent characterization of 4′OMT from E. californica revealed low 6OMT activity, suggesting a role for 4′OMT as a surrogate for 6OMT to facilitate BIA biosynthesis.

Metabolic engineering of MIA biosynthesis in C. roseus

Plant cell culture technology has been promoted as a platform for the commercial production of useful secondary metabolites. The large-scale production of shikonin (Tabata, 1996), berberine (Zhao and Verpoorte, 2007), ginseng saponins (Moyano et al., 2005) and Taxol (Tabata, 2004) are key examples. Despite these successes, the many failures have been attributed to various biological limitations resulting in low or unstable production of interesting metabolites (Zhao and Verpoorte, 2007). This has been particularly true in the case of C. roseus cell cultures, especially in terms of the inability of cultured cells to produce vindoline. As a result of the present availability of numerous MIA biosynthetic and regulatory genes, several engineering efforts to modify alkaloid metabolism in transgenic C. roseus cell and root cultures have been reported (Pasquali et al., 2006; Zárate and Verpoorte, 2007; Zhao and Verpoorte, 2007).

Transgenic C. roseus cell cultures over-expressing TDC and/or STR1 did not produce higher levels of MIAs compared with controls (Canel et al., 1998; Whitmer et al., 1998). Further experimentation suggested limited precursor availability as supplementation with loganin and tryptamine facilitated an increase in alkaloid accumulation. A number of other studies showed that the simple over-expression of individual genes in the MIA pathway was insufficient to increase stable alkaloid production in the absense of an increase in the production of organized tissues, such as hairy roots (Pasquali et al., 2006). For example, expression of ORCA3 in cell cultures activated the expression of tryptophan biosynthetic genes, as well as of the gene encoding tryptophan decarboxylase. Although tryptamine accumulation increased, the transgenic cell cultures remained limited in their ability to produce monoterpenoid precursors, such as loganin (van der Fits and Memelink, 2000). The potential of hairy root cultures for the stable production of alkaloids and other secondary metabolites has been attributed to a higher degree of genetic and biochemical stability due to increased cellular differentiation compared with cell cultures (Pasquali et al., 2006; Shanks et al., 1998; Zárate and Verpoorte, 2007; Zhao and Verpoorte, 2007).

In recent studies, C. roseus hairy root cultures expressing various forms of anthranilate synthase and/or TDC exhibited higher flux through the tryptophan branch of the MIA pathway (Hughes et al., 2004). Hairy root cultures expressing these genes under the control of constitutive or inducible promoters accumulated higher levels of tryptamine rather than alkaloids. Other attempts to alter the isoprenoid pathway by expressing a hamster 3-hydroxy-3-methylglutaryl CoA reductase gene in hairy roots generated one line that accumulated up to sevenfold higher levels of serpentine compared with controls (Ayora-Talavera et al., 2002). However, no effects on catharanthine levels were detected. Hairy root cultures over-expressing DAT resulted in several lines that accumulated up to fourfold higher levels of hörhammericine compared with controls (Magnotta et al., 2007). To explain these results, evidence was provided to show that an interaction occurring between DAT and the root-specific MAT inhibited conversion of hörhammericine to 19-O-acetylhörhammericine. Studies with inhibitors showed that MIAs turn over during the growth of hairy root cultures (Morgan and Shanks, 1999). Together these results provided a plausible explanation for the accumulation of hörhammericine, assuming that the O-acetylation step is required for turnover of tabersonine, lochnericine and hörhammericine.

The potential for enhancing the MIA content of C. roseus plants has not been analyzed in detail. Although a few reports have addressed whole-plant metabolic engineering (Zárate and Verpoorte, 2007), the use of traditional breeding to improve the yield of MIAs has not been emphasized. Analysis of a variety of C. roseus germplasms suggests that mutation breeding could represent a source of high-MIA genotypes (Dutta et al., 2005; Magnotta et al., 2006).

Metabolic engineering of substrate specificity of MIA pathway enzymes

The molecular architecture of major enzymes in the ajmaline pathway (Figure 2) has recently been reviewed (Stöckigt et al., 2007). Several refined [STR (Ma et al., 2006) and SGD (Barleben et al., 2007)] and partial [raucaffricine glucosidase (Ruppert et al., 2006), perakine reductase (Rosenthal et al., 2006) and vinorine synthase (Ma et al., 2005)] crystal structures for enzymes involved in MIA biosynthesis in R. serpentina have recently been reported (Figure 2). The information obtained from the crystal structure of STR complexed with strictosidine was used to produce the rational site-directed V208A mutation of STR. The mutant STR accepted a larger range of indoleamine and iridoid substrates (Loris et al., 2007). When mutant STR was coupled to SDG from C. roseus, a large alkaloid library for pharmacological screening and novel drug design was generated. Similarly, the STR mutant D177A increased the turnover of certain secologanin analogs (Chen et al., 2006) compared with wild-type STR (McCoy and O’Connor, 2006). The crystal structure of Rserpentina STS complexed with tryptamine (Ma et al., 2006) was also used in the rational generation of two site-directed V214M and F232L mutants able to produce several analogs of strictosidine with various substituents (F, Br or CH3OH) (Bernhardt et al., 2007). When analogs of 5-(R)-hydroxymethyl strictosidine or 10-bromostrictosidine were fed to C. roseus hairy root cultures, a number of putative modified products were isolated.

Outlook

  1. Top of page
  2. Summary
  3. Introduction
  4. Benzylisoquinoline alkaloid metabolism
  5. Monoterpenoid indole alkaloid metabolism
  6. The role of ‘omics’ technologies to study plant alkaloid biosynthesis
  7. Compartmentalization and trafficking
  8. Gene regulation and signal transduction
  9. Metabolic engineering of MIA and BIA biosynthesis
  10. Outlook
  11. Acknowledgements
  12. References

Opium poppy and C. roseus have emerged as premier model, non-model systems to investigate the biochemistry and biology of alkaloid metabolism in plants. Molecular clones encoding an impressive number of BIA and MIA biosynthetic enzymes have been isolated, and the pace of gene discovery is increasing with the application of ‘omics’ technologies. EST databases and DNA microarray chips are proven tools in these systems, and proteomics and metabolomics platforms are steadily improving. In opium poppy, functional genomic strategies have benefitted from the establishment of genetic transformation protocols, the development of new techniques such as TILLING (Comai and Henikoff, 2006), and the wealth of genetic diversity obtained through intensive classical breeding. Although transformed root cultures are useful for metabolic engineering, the full potential of functional genomics in C. roseus remains restricted by the present inability to transform and regenerate plants. The possibility of reconstituting the complex metabolic pathways leading to morphine, vinblastine and other pharmaceutical alkaloids in heterologous organisms, such as yeast and bacteria, is approaching feasibility as the number of biosynthetic genes rapidly increases.

Future research on the regulation of BIA and MIA biosynthesis should continue to target the isolation of transcription factors involved in the coordinated regulation of pathway enzymes and other metabolic components. The intra- and inter-cellular translocation of pathway intermediates and products, which might involve specific transporters or the symplastic movement of metabolites, is another important topic that requires attention. The complex subcellular organization of the biosynthetic machinery involved in BIA and MIA metabolism is apparent, but remains poorly understood. A thorough understanding of metabolic regulation – at the transcriptional, cellular and biochemical levels – is crucial to realize the biotechnological goal of rationally engineering alkaloid metabolism in opium poppy and C. roseus.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Benzylisoquinoline alkaloid metabolism
  5. Monoterpenoid indole alkaloid metabolism
  6. The role of ‘omics’ technologies to study plant alkaloid biosynthesis
  7. Compartmentalization and trafficking
  8. Gene regulation and signal transduction
  9. Metabolic engineering of MIA and BIA biosynthesis
  10. Outlook
  11. Acknowledgements
  12. References

Research in the Facchini and De Luca laboratories has been funded by Discovery and Strategic Project grants from the Natural Sciences and Engineering Research Council of Canada. P.J.F. and V.D.L. also gratefully acknowledge the NAPGEN Genomics Resource Consortium established by the National Research Council–Plant Biotechnology Institute (Saskatoon, Saskatchewan, Canada). P.J.F. holds the Canada Research Chair in Plant Metabolic Processes Biotechnology. V.D.L. holds the Canada Research Chair in Plant Biochemistry and Biotechnology. We thank Jillian Hagel (University of Calgary, Canada) for assistance with the anatomical drawings and Jun Murata (Nara Institute of Technology, Japan) for providing the C. roseus leaf cross-section.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Benzylisoquinoline alkaloid metabolism
  5. Monoterpenoid indole alkaloid metabolism
  6. The role of ‘omics’ technologies to study plant alkaloid biosynthesis
  7. Compartmentalization and trafficking
  8. Gene regulation and signal transduction
  9. Metabolic engineering of MIA and BIA biosynthesis
  10. Outlook
  11. Acknowledgements
  12. References
  • Alcantara, J., Bird, D.A., Franceschi, V.R. and Facchini, P.J. (2005) Sanguinarine biosynthesis is associated with the endoplasmic reticulum in cultured opium poppy cells after elicitor treatment. Plant Physiol. 138, 173183.
  • Allen, R.S., Millgate, A.G., Chitty, J.A., Thisleton, J., Miller, J.A., Fist, A.J., Gerlach, W.L. and Larkin, P.J. (2004) RNAi-mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy. Nat. Biotechnol. 22, 15591566.
  • Allen, R.S., Miller, J.A., Chitty, J.A., Fist, A.J., Gerlach, W.L. and Larkin, P.J. (2008) Metabolic engineering of morphinan alkaloids by over-expression and RNAi suppression of salutaridinol 7-O-acetyltransferase in opium poppy. Plant Biotechnol. J., 6, 2230.
  • Amann, M., Wanner, G. and Zenk, M.H. (1986) Intracellular compartmentation of two enzymes of berberine biosynthesis in plant cell cultures. Planta, 167, 310320.
  • Ayora-Talavera, T., Chappell, J., Lozoya-Gloria, E. and Loyola-Vargas, V.M. (2002) Overexpression in Catharanthus roseus hairy roots of a truncated hamster 3-hydroxy-3-methylglutaryl-CoA reductase gene. Appl. Biochem. Biotechnol. 97, 135145.
  • Barleben, L., Panjikar, S., Ruppert, M., Koepke, J. and Stöckigt, J. (2007) Molecular architecture of strictosidine glucosidase: the gateway to the biosynthesis of the monoterpenoid indole alkaloid family. Plant Cell, 19, 28862897.
  • Bauer, W. and Zenk, M.H. (1991) Two methylenedioxy bridge forming cytochrome P-450 dependent enzymes are involved in (S)-stylopine biosynthesis. Phytochemistry, 30, 29532961.
  • Belny, M., Hérouart, D., Thomasset, B., David, H., Jacquin-Dubreuil, A. and David, A. (1997) Transformation of Papaver somniferum cell suspension cultures with sam1 from A. thaliana results in cell lines of different S-adenosyl-l-methionine synthetase activity. Physiol. Plant. 99, 233240.
  • Bernhardt, P., McCoy, E. and O’Connor, S.E. (2007) Rapid identification of enzyme variants for reengineered alkaloid biosynthesis in periwinkle. Chem. Biol. 14, 888897.
  • 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, 888897.
  • Bird, D.A., Franceschi, V.R. and Facchini, P.J. (2003) A tale of three cell types: alkaloid biosynthesis is localized to sieve elements in opium poppy. Plant Cell, 15, 26262635.
  • Burlat, V., Oudin, A., Courtois, M., Rideau, M. and St-Pierre, B. (2004) Co-expression of three MEP pathway genes and geraniol 10-hydroxylase in internal phloem parenchyma of Catharanthus roseus implicates multicellular translocation of intermediates during the biosynthesis of monoterpene indole alkaloids and isoprenoid-derived primary metabolites. Plant J. 38, 131141.
  • Cacacea, S., Schröder, G., Wehingera, E., Strack, D., Schmidt, J. and Schröder, J. (2003) A flavonol O-methyltransferase from Catharanthus roseus. Phytochemistry, 62, 127137.
  • Canel, C., Lopes-Cardoso, I., Whitmer, S., Van Der Fits, L., Pasquali, G., Van Der Heijden, R., Hoge, J.H.C. and Verpoorte, R. (1998) Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus. Planta, 205, 414419.
  • Carlson, J.E., Leebens-Mack, J.H., Wall, P.K. et al. (2006) EST database for early flower development in California poppy (Eschscholzia californica Cham., Papaveraceae) tags over 6000 genes from a basal eudicot. Plant Mol. Biol. 62, 351369.
  • Chahed, K., Oudin, A., Guivarc’h, N., Hamdi, S., Chénieux, J.C., Rideau, M. and Clastre, M. (2000) 1-Deoxy-d-xylulose 5-phosphate synthase from periwinkle: cDNA identification and induced gene expression in terpenoid indole alkaloid-producing cells. Plant Physiol. Biochem. 38, 559566.
  • Chen, S., Galan, M.C., Coltharp, C. and O’Connor, S.E. (2006) Redesign of a central enzyme in alkaloid biosynthesis. Chem. Biol. 13, 11371141.
  • Chitty, J.A., Allen, R.S., Fist, A.J. and Larkin, P.J. (2003) Genetic transformation in commercial Tasmanian cultivars of opium poppy, Papaver somniferum, and movement of transgenic pollen in the field. Funct. Plant Biol. 30, 10451058.
  • Choi, K.B., Morishige, T., Shitan, N., Yazaki, K. and Sato, F. (2002) Molecular cloning and characterization of coclaurine N-methyltransferase from cultured cells of Coptis japonica. J. Biol. Chem. 277, 830835.
  • Choi, Y.H., Tapias, E.C., Kim, H.K., Lefeber, A.W., Erkelens, C., Verhoeven, J.T., Brzin, J., Zel, J. and Verpoorte, R. (2004) Metabolic discrimination of Catharanthus roseus leaves infected by phytoplasma using 1H-NMR spectroscopy and multivariate data analysis. Plant Physiol. 135, 23982410.
  • Collu, G., Unver, N., Peltenburg-Looman, A.M., Van Der Heijden, R., Verpoorte, R. and Memelink, J. (2001) Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis. FEBS Lett. 508, 215220.
  • Comai, L. and Henikoff, S. (2006) TILLING: practical single nucleotide mutation discovery. Plant J. 45, 684694.
  • Contin, A., Van Der Heijden, R., Lefeber, A.W. and Verpoorte, R. (1998) The iridoid glucoside secologanin is derived from the novel triose phosphate/pyruvate pathway in a Catharanthus roseus cell culture. FEBS Lett. 434, 413416.
  • Courdavault, V., Thiersault, M., Courtois, M., Gantet, P., Oudin, A., Doireau, P., St-Pierre, B. and Giglioli-Guivarc’h, N. (2005) CaaX-prenyltransferases are essential for expression of genes involved in the early stages of monoterpenoid biosynthetic pathway in Catharanthus roseus cells. Plant Mol. Biol. 57, 855870.
  • De Luca, V. and Cutler, A.J. (1987) Subcellular localization of enzymes involved in indole alkaloid biosynthesis in Catharanthus roseus. Plant Physiol. 85, 10991102.
  • De Luca, V., Marineau, C. and Brisson, N. (1989) Molecular cloning and analysis of cDNA encoding a plant tryptophan decarboxylase: comparison with animal dopa decarboxylases. Proc. Natl Acad. Sci. USA, 86, 25822586.
  • Decker, G., Wanner, G., Zenk, M.H. and Lottspeich, F. (2000) Characterization of proteins in latex of the opium poppy (Papaver somniferum) using two-dimensional gel electrophoresis and microsequencing. Electrophoresis, 21, 35003516.
  • De-Eknamkul, W. and Zenk, M.H. (1992) Purification and properties of 1,2-dehydroreticuline reductase from Papaver somniferum seedlings. Phytochemistry, 31, 813821.
  • Dethier, M. and De Luca, V. (1993) Partial purification of an N-methyltransferase involved in vindoline biosynthesis in Catharanthus roseus. Phytochemistry, 32, 673678.
  • Dittrich, H. and Kutchan, T.M. (1991) Molecular cloning, expression, and induction of berberine bridge enzyme, an enzyme essential to the formation of benzophenanthridine alkaloids in the response of plants to pathogenic attack. Proc. Natl Acad. Sci. USA, 88, 99699973.
  • Dutta, A., Batra, J., Pandey-Rai, S., Singh, D., Kumar, S. and Sen, J. (2005) Expression of terpenoid indole alkaloid biosynthetic pathway genes corresponds to accumulation of related alkaloids in Catharanthus roseus (L.) G. Don. Planta, 220, 376383.
  • El-Sayed, M. and Verpoorte, R. (2007) Catharanthus terpenoid indole alkaloids: biosynthesis and regulation. Phytochem. Rev. 6, 277305.
  • Facchini, P.J. (2006) Regulation of alkaloid biosynthesis in plants. Alkaloids Chem. Biol. 63, 144.
  • Facchini, P.J. and De Luca, V. (1994) Differential and tissue-specific expression of a gene family for tyrosine/dopa decarboxylase in opium poppy. J. Biol. Chem. 269, 2668426690.
  • Facchini, P.J. and De Luca, V. (1995) Phloem-specific expression of tyrosine/dopa decarboxylase genes and the biosynthesis of isoquinoline alkaloids in opium poppy. Plant Cell, 7, 18111821.
  • Facchini, P.J. and Park, S.U. (2003) Developmental and inducible accumulation of gene transcripts involved in alkaloid biosynthesis in opium poppy. Phytochemistry, 64, 177186.
  • Facchini, P.J., Penzes, C., Johnson, A.G. and Bull, D. (1996) Molecular characterization of berberine bridge enzyme genes from opium poppy. Plant Physiol. 112, 16691677.
  • Facchini, P.J., Loukanina, N. and Blanche, V. (2008) Genetic transformation via somatic embryogenesis to establish herbicide-resistant opium poppy. Plant Cell Rep., in press.
  • Färber, K., Schumann, B., Miersch, O. and Roos, W. (2003) Selective desensitization of jasmonate- and pH-dependent signaling in the induction of benzophenanthridine biosynthesis in cells of Eschscholzia californica. Phytochemistry, 62, 491500.
  • Van Der Fits, L. and Memelink, J. (2000) ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science, 289, 295297.
  • Van Der Fits, L., Zhang, H., Menke, F.L.H., Deneka, M. and Memelink, J. (2000) A Catharanthus roseus BPF-1 homologue interacts with an elicitor-responsive region of the secondary metabolite biosynthetic gene Str and is induced by elicitor via a jasmonate-independent signal transduction pathway. Plant Mol. Biol. 44, 675685.
  • Van Der Fits, L., Hilliou, F. and Memelink, J. (2001) T-DNA activation tagging as a tool to isolate regulators of a metabolic pathway from a genetically non-tractable plant species. Transgenic Res. 10, 513521.
  • Frederich, M., Choi, Y.H., Angenot, L., Harnischfeger, G., Lefeber, A.W.M. and Verpoorte, R. (2004) Metabolomic analysis of Strychnos nux-vomica, Strychnos icaja and Strychnos ignatii extracts by 1H nuclear magnetic resonance spectrometry and multivariate analysis techniques. Phytochemistry, 65, 19932001.
  • Frick, S., Chitty, J.A., Kramell, R., Schmidt, J., Allen, R.S., Larkin, P.J. and Kutchan, T.M. (2004) Transformation of opium poppy (Papaver somniferum L.) with antisense berberine bridge enzyme gene (anti-bbe) via somatic embryogenesis results in an altered ratio of alkaloids in latex but not in roots. Transgenic Res. 13, 607613.
  • Frick, S., Kramell, R. and Kutchan, T.M. (2007) Metabolic engineering with a morphine biosynthetic P450 in opium poppy surpasses breeding. Metab. Eng. 9, 169176.
  • Fujii, N., Inui, T., Iwasa, K., Morishige, T. and Sato, F. (2007) Knockdown of berberine bridge enzyme by RNAi accumulates (S)-reticuline and activates a silent pathway in cultured California poppy cells. Transgenic Res. 16, 363375.
  • Gantet, P., Imbault, N., Thiersault, M. and Doireau, P. (1998) Necessity of a functional octadecanoic pathway for indole alkaloid synthesis by Catharanthus roseus cell suspensions cultured in an auxin-starved medium. Plant Cell Physiol. 39, 220225.
  • Geerlings, A., Ibanez, M.M., Memelink, J., Van Der Heijden, R. and Verpoorte, R. (2000) Molecular cloning and analysis of strictosidine β-d-glucosidase, an enzyme in terpenoid indole alkaloid biosynthesis in Catharanthus roseus. J. Biol. Chem. 275, 30513056.
  • Gerardy, R. and Zenk, M.H. (1993a) Formation of salutaridine from (R)-reticuline by a membrane-bound cytochrome P-450 enzyme from Papaver somniferum. Phytochemistry, 32, 7986.
  • Gerardy, R. and Zenk, M.H. (1993b) Purification and characterization of salutaridine:NADPH 7-oxidoreductase from Papaver somniferum. Phytochemistry, 34, 125132.
  • Goossens, A. and Rischer, H. (2007) Implementation of functional genomics for gene discovery in alkaloid producing plants. Phytochem. Rev. 6, 3549.
  • Goossens, A., Häkkinen, S.T., Laakso, I. et al. (2003) A functional genomics approach toward the understanding of secondary metabolism in plant cells. Proc. Natl Acad. Sci. USA, 100, 85958600.
  • Grothe, T., Lenz, R. and Kutchan, T.M. (2001) Molecular characterization of the salutaridinol 7-O-acetyltransferase involved in morphine biosynthesis in opium poppy Papaver somniferum. J. Biol. Chem. 276, 3071730723.
  • Hancock, R.D., McRae, D., Haupt, S. and Viola, R. (2003) Synthesis of l-ascorbic acid in the phloem. BMC Plant Biol. 3, 7.
  • Hause, B., Hause, G., Kutter, C., Miersch, O. and Wasternack, C. (2003) Enzymes of jasmonate biosynthesis occur in tomato sieve elements. Plant Cell Physiol. 44, 643648.
  • Hedhili, S., Courdavault, V., Giglioli-Guivarc’h, N. and Gantet, P. (2007) Regulation of the terpene moiety biosynthesis of Catharanthus roseus terpene indole alkaloids. Phytochem. Rev. 6, 341351.
  • Van Der Heijden, R., Jacobs, D.I., Snoeijer, W., Hallard, D. and Verpoorte, R. (2004) The Catharanthus alkaloids: pharmacognosy and biotechnology. Curr. Med. Chem. 11, 607628.
  • Heinze, M., Steighardt, J., Gesell, A., Schwartze, W. and Roos, W. (2007) Regulatory interaction of the Gα protein with phospholipase A(2) in the plasma membrane of Eschscholzia californica. Plant J. 52, 10411051.
  • Hibi, N., Higashiguchi, S., Hashimoto, T. and Yamada, Y. (1994) Gene expression in tobacco low-nicotine mutants. Plant Cell, 6, 723735.
  • Hirata, K., Horiuchi, M., Ando, T., Miyamoto, K. and Miura, Y. (1990) Vindoline and catharanthine production in multiple shoot cultures of Catharanthus roseus. J. Ferment. Bioeng. 70, 193195.
  • Hirata, K., Poeaknapo, C., Schmidt, J. and Zenk, M.H. (2004) 1,2-Dehydroreticuline synthase, the branch point enzyme opening the morphinan biosynthetic pathway. Phytochemistry, 65, 10391046.
  • Hisiger, S. and Jolicoeur, M. (2007) Analysis of Catharanthus roseus alkaloids by HPLC. Phytochem. Rev. 6, 207234.
  • Huang, F.C. and Kutchan, T.M. (2000) Distribution of morphinan and benzo[c]phenanthridine alkaloid gene transcript accumulation in Papaver somniferum. Phytochemistry, 53, 555564.
  • Hughes, E.H., Hong, S.B., Gibson, S.I., Shanks, J.V. and San, K.Y. (2004) Metabolic engineering of the indole pathway in Catharanthus roseus hairy roots and increased accumulation of tryptamine and serpentine. Metab. Eng. 6, 268276.
  • Ikeda, H., Esaki, N., Nakai, S., Hashimoto, K., Uesato, S., Soda, K. and Fujita, T. (1991) Acyclic monoterpene primary alcohol:NADP+ oxidoreductase of Rauwolfia serpentina cells: the key enzyme in biosynthesis of monoterpene alcohols. J. Biochem. 109, 341347.
  • Ikezawa, N., Tanaka, M., Nagayoshi, M., Shinkyo, R., Sakaki, T., Inouye, K. and Sato, F. (2003) Molecular cloning and characterization of CYP719, a methylenedioxy bridge-forming enzyme that belongs to a novel P450 family, from cultured Coptis japonica cells. J. Biol. Chem. 278, 3855738565.
  • Ikezawa, N., Iwasa, K. and Sato, F. (2007) Molecular cloning and characterization of methylenedioxy bridge-forming enzymes involved in stylopine biosynthesis in Eschscholzia californica. FEBS J. 274, 10191035.
  • Inui, T., Tamura, K., Fujii, N., Morishige, T. and Sato, F. (2007) Overexpression of Coptis japonica norcoclaurine 6-O-methyltransferase overcomes the rate-limiting step in benzylisoquinoline alkaloid biosynthesis in cultured Eschscholzia californica. Plant Cell Physiol. 48, 252262.
  • Irmler, S., Schröder, G., St. Pierre, B., Crouch, N.P., Hotze, M., Schmidt, J., Strack, D., Mattern, U. and Schröder, J. (2000) Indole alkaloid biosynthesis in Catharanthus roseus: new enzyme activities and identification of cytochrome P450 CYP72A2 as secologanin synthase. Plant J. 24, 797804.
  • Jacobs, D.I., Gaspari, M., Van Der Greef, J., Van Der Heijden, R. and Verpoorte, R. (2005) Proteome analysis of the medicinal plant Catharanthus roseus. Planta, 221, 690704.
  • Jorgensen, R.A., Atkinson, R.G., Forster, R.L. and Lucas, W.J. (1998) An RNA-based information superhighway in plants. Science, 279, 14861487.
  • Kato, N., Dubouzet, E., Kokabu, Y., Yoshida, S., Taniguchi, Y., Yazaki, K. and Sato, F. (2007) Identification of a WRKY protein as a transcriptional regulator of benzylisoquinoline alkaloid biosynthesis in Coptis japonica. Plant Cell Physiol. 48, 818.
  • Kraus, P.F. and Kutchan, T.M. (1995) Molecular cloning and heterologous expression of a cDNA encoding berbamunine synthase, a C–O phenol-coupling cytochrome P450 from the higher plant Berberis stolonifera. Proc. Natl Acad. Sci. USA, 92, 20712075.
  • Kutchan, T.M., Hampp, N., Lottspeich, F., Beyreuther, K. and Zenk, M.H. (1988) The cDNA clone for strictosidine synthase from Rauvolfia serpentina. DNA sequence determination and expression in Escherichia coli. FEBS Lett. 237, 4044.
  • Laflamme, P., St-Pierre, B. and De Luca, V. (2001) Molecular and biochemical analysis of a Madagascar periwinkle root-specific minovincinine-19-hydroxy-O-acetyltransferase. Plant Physiol. 125, 189198.
  • Larkin, P. and Harrigan, G.G. (2007) Opportunities and surprises in crops modified by transgenic technology: metabolic engineering of benzylisoquinoline alkaloid, gossypol and lysine biosynthetic pathways. Metabolomics, 3, 371382.
  • Larkin, P.J., Miller, J.A., Allen, R.S., Chitty, J.A., Gerlach, W.L., Frick, S., Kutchan, T.M. and Fist, A.J. (2007) Increasing morphinan alkaloid production by over-expressing codeinone reductase in transgenic Papaver somniferum. Plant Biotechnol. J. 5, 2637.
  • Le Flem-Bonhomme, V., Laurain-Mattar, D. and Fliniaux, M.A. (2004) Hairy root induction of Papaver somniferum var. album, a difficult-to-transform plant, by A. rhizogenes LBA 9402. Planta, 218, 890893.
  • Levac, D., Murata, J., Kim, W.S. and De Luca, V. (2008) Application of carborundum abrasion for investigating leaf epidermis: molecular cloning of Catharanthus roseus 16-hydroxytabersonine-16-O-methyltransferase. Plant J. 53, 225236.
  • Liscombe, D.K. and Facchini, P.J. (2007) Molecular cloning and characterization of tetrahydroprotoberberine cis-N-methyltransferase, an enzyme involved in alkaloid biosynthesis in opium poppy. J. Biol. Chem. 282, 1474114751.
  • Liscombe, D.K., MacLeod, B.P., Loukanina, N., Nandi, O.I. and Facchini, P.J. (2005) Evidence for the monophyletic evolution of benzylisoquinoline alkaloid biosynthesis in angiosperms. Phytochemistry, 66, 25012520.
  • Loris, E.A., Panjikar, S., Ruppert, M., Barleben, L., Unger, M., Schuebel, H. and Stöckigt, J. (2007) Structure-based engineering of strictosidine synthase: auxiliary for alkaloid libraries. Chem. Biol. 14, 979985.
  • Loyola-Vargas, V.M., Galaz-Ávalos, R.M. and Kú-Cauich, R. (2007) Catharanthus biosynthetic enzymes: the road ahead. Phytochem. Rev. 6, 307339.
  • Luk, L.Y., Bunn, S., Liscombe, D.K., Facchini, P.J. and Tanner, M.E. (2007) Mechanistic studies on norcoclaurine synthase of benzylisoquinoline alkaloid biosynthesis: an enzymatic Pictet–Spengler reaction. Biochemistry, 46, 1015310161.
  • Ma, X., Koepke, J., Bayer, A., Fritzsch, G., Michel, H. and Stöckigt, J. (2005) Crystallization and preliminary X-ray analysis of native and selenomethionyl vinorine synthase from Rauvolfia serpentina. Acta Crystallogr. D Biol. Crystallogr. 61, 694696.
  • Ma, X., Panjikar, S., Koepke, J., Loris, E. and Stöckigt, J. (2006) The structure of Rauvolfia serpentina strictosidine synthase is a novel six-bladed beta-propeller fold in plant proteins. Plant Cell, 18, 907920.
  • Madyastha, K.M., Guarnaccia, R., Baxter, C. and Coscia, C.J. (1973) S-Adenosyl-l-methionine: loganic acid methyltransferase. J. Biol. Chem. 248, 24972501.
  • Magnotta, M., Murata, J., Chen, J. and De Luca, V. (2006) Identification of a low vindoline accumulating cultivar of Catharanthus roseus (L.) G. Don by alkaloid and enzymatic profiling. Phytochemistry, 67, 17581764.
  • Magnotta, M., Murata, J., Chen, J. and De Luca, V. (2007) Expression of deacetylvindoline-4-O-acetyltransferase in Catharanthus roseus hairy roots. Phytochemistry, 68, 19221931.
  • Mahroug, S., Courdavault, V., Thiersault, M., St-Pierre, B. and Burlat, V. (2006) Epidermis is a pivotal site of at least four secondary metabolic pathways in Catharanthus roseus aerial organs. Planta, 223, 11911200.
  • Mahroug, S., Burlat, V. and St-Pierre, B. (2007) Cellular and sub-cellular organisation of the monoterpenoid indole alkaloid pathway in Catharanthus roseus. Phytochem. Rev. 6, 363381.
  • Maldonado-Mendoza, I.E., Burnett, R.J. and Nessler, C.L. (1992) Nucleotide sequence of a cDNA encoding 3-hydroxy-3-methylglutaryl-CoA reductase from Catharanthus roseus. Plant Physiol. 100, 16131614.
  • McCoy, E. and O’Connor, S.E. (2006) Directed biosynthesis of alkaloid analogs in the medicinal plant Catharanthus roseus. J. Am. Chem. Soc. 128, 1427614277.
  • McKnight, T.D., Roessner, C.A., Devagupta, R., Scott, A.I. and Nessler, C.L. (1990) Nucleotide sequence of a cDNA encoding the vacuolar protein strictosidine synthase from Catharanthus roseus. Nucleic Acids Res. 18, 4939.
  • McKnight, T.D., Bergey, D.R., Burnett, R.J. and Nessler, C.L. (1991) Expression of enzymatically active and correctly targeted strictosidine synthase in transgenic tobacco plants. Planta, 185, 148152.
  • Memelink, J. and Gantet, P. (2007) Transcription factors involved in terpenoid indole alkaloid biosynthesis in Catharanthus roseus. Phytochem. Rev. 6, 353362.
  • Menke, F.L.H., Champion, A., Kijne, J.W. and Memelink, J. (1999) A novel jasmonate- and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a jasmonate- and elicitor inducible AP2-domain transcription factor, ORCA2. EMBO J. 18, 44554463.
  • Millgate, A.G., Pogson, B.J., Wilson, I.W., Kutchan, T.M., Zenk, M.H., Gerlach, W.L., Fist, J. and Larkin, P.J. (2004) Analgesia: morphine-pathway block in top1 poppies. Nature, 431, 413414.
  • Minami, H., Dubouzet, E., Iwasa, K. and Sato, F. (2007) Functional analysis of norcoclaurine synthase in Coptis japonica. J. Biol. Chem. 282, 62746282.
  • Morgan, J. and Shanks, J. (1999) Inhibitor studies of tabersonine metabolism in C. roseus hairy roots. Phytochemistry, 51, 6168.
  • Morishige, T., Tsujita, T., Yamada, Y. and Sato, F. (2000) Molecular characterization of the S-adenosyl-l-methionine:3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase involved in isoquinoline alkaloid biosynthesis in Coptis japonica. J. Biol. Chem. 275, 2339823405.
  • Morishige, T., Dubouzet, E., Choi, K.B., Yazaki, K. and Sato, F. (2002) Molecular cloning of columbamine O-methyltransferase from cultured Coptis japonica cells. Eur. J. Biochem. 269, 56595667.
  • Moyano, E., Osuna, L., Bonfill, M., Cusido, R.M., Palazon, J., Tortoriello, J. and Pinol, M.T. (2005) Bioproduction of triterpenes on plant cell cultures of Panax ginseng and Galphimia glauca. Rec. Res. Dev. Plant Sci. 3, 195213.
  • Murata, J. and De Luca, V. (2005) Localization of tabersonine 16-hydroxylase and 16-OH tabersonine-16-O-methyltransferase to leaf epidermal cells defines them as a major site of precursor biosynthesis in the vindoline pathway in Catharanthus roseus. Plant J. 44, 581594.
  • Murata, J., Bienzle, D., Brandle, J.E., Sensen, C.W. and De Luca, V. (2006) Expressed sequence tags from Madagascar periwinkle (Catharanthus roseus). FEBS Lett. 580, 45014507.
  • Murata, J., Roepke, J., Gordon, H. and De Luca, V. (2008) ‘Surfaceome’ analysis of leaf epidermal cells in Catharanthus roseus reveals its biochemical specialization. Plant Cell, doi: DOI: 10.1105/tpc.107.056630.
  • O’Connor, S.E. and Maresh, J.M. (2006) Chemistry and biology of terpene indole alkaloid biosynthesis. Nat. Prod. Rep. 23, 532547.
  • Oudin, A., Courtois, M., Rideau, M. and Clastre, M. (2007a) The iridoid pathway in Catharanthus roseus alkaloid biosynthesis. Phytochem. Rev. 6, 259276.
  • Oudin, A., Mahroug, S., Courdavault, V., Hervouet, N., Zelwer, C., Rodriguez-Concepcion, M., St-Pierre, B. and Burlat, V. (2007b) Spatial distribution and hormonal regulation of gene products from methyl erythritol phosphate and monoterpene-secoiridoid pathways in Catharanthus roseus. Plant Mol. Biol. 65, 1330.
  • Ounaroon, A., Decker, G., Schmidt, J., Lottspeich, F. and Kutchan, T.M. (2003) (R,S)-Reticuline 7-O-methyltransferase and (R,S)-norcoclaurine 6-O-methyltransferase of Papaver somniferum– cDNA cloning and characterization of methyl transfer enzymes of alkaloid biosynthesis in opium poppy. Plant J. 36, 808819.
  • Ouwerkerk, P.B.F. and Memelink, J. (1999) A G-box element from the Catharanthus roseus strictosidine synthase (Str) gene promoter confers seed-specific expression in transgenic tobacco plants. Mol. Gen. Genet. 261, 635643.
  • Park, S.U. and Facchini, P.J. (2000a) Agrobacterium-mediated transformation of opium poppy, Papaver somniferum L., via shoot organogenesis. J. Plant Physiol. 157, 207214.
  • Park, S.U. and Facchini, P.J. (2000b) Agrobacterium rhizogenes-mediated transformation of opium poppy, Papaver somniferum L., and California poppy, Eschscholzia californica Cham., root cultures. J. Exp. Bot. 51, 10051016.
  • Park, S.U., Yu, M. and Facchini, P.J. (2002) Antisense RNA-mediated suppression of benzophenanthridine alkaloid biosynthesis in transgenic cell cultures of California poppy. Plant Physiol. 128, 696706.
  • Park, S.U., Yu, M. and Facchini, P.J. (2003) Modulation of berberine bridge enzyme levels in transgenic root cultures of California poppy alters the accumulation of benzophenanthridine alkaloids. Plant Mol. Biol. 51, 153164.
  • Pasquali, G., Porto, D.D. and Fett-Neto, A.G. (2006) Metabolic engineering of cell cultures versus whole plant complexity in production of bioactive monoterpene indole alkaloids: recent progress related to an old dilemma. J. Biosci. Bioeng. 101, 287296.
  • Pauli, H.H. and Kutchan, T.M. (1998) Molecular cloning and functional heterologous expression of two alleles encoding (S)-N-methylcoclaurine 3′-hydroxylase (CYP80B1), a new methyl jasmonate-inducible cytochrome P-450-dependent monooxygenase of benzylisoquinoline alkaloid biosynthesis. Plant J. 13, 793801.
  • Pauw, B., Hilliou, F.A.O., Sandonis, M.V. et al. (2004) Zinc finger proteins act as transcriptional repressors of alkaloid biosynthesis genes in Catharanthus roseus. J. Biol. Chem. 279, 5294052948.
  • Pilatzke-Wunderlich, I. and Nessler, C.L. (2001) Expression and activity of cell-wall-degrading enzymes in the latex of opium poppy, Papaver somniferum L. Plant Mol. Biol. 45, 567576.
  • Rischer, H., Oreši, M., Seppänen-Laakso, T., Katajamaa, M., Lammertyn, F., Ardiles-Diaz, W., Van Montagu, M.C.E., Inzé, D., Oksman-Caldentey, K.M. and Goossens, A. (2006) Gene-to-metabolite networks for terpenoid indole alkaloid biosynthesis in Catharanthus roseus cells. Proc. Natl Acad. Sci. USA, 103, 56145619.
  • Rodriguez, S., Compagnon, V., Crouch, N.P., St Pierre, B. and De Luca, V. (2003) Jasmonate-induced epoxidation of tabersonine by cytochrome P-450 in hairy root cultures of Catharanthus roseus. Phytochemistry, 64, 401409.
  • Rohmer, M. (1999) The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants. Nat. Prod. Rep. 16, 565574.
  • Rosenthal, C., Mueller, U., Panjikar, S., Sun, L., Ruppert, M., Zhao, Y. and Stöckigt, J. (2006) Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 62, 12861289.
  • Roytrakul, S. and Verpoorte, R. (2007) Role of vacuolar transporter proteins in plant secondary metabolism: Catharanthus roseus cell culture. Phytochem. Rev. 6, 383396.
  • Rueffer, M. and Zenk, M.H. (1987) Enzymatic formation of protopines by a microsomal cytochrome P-450 system of Corydalis vaginans. Tetrahedron Lett. 28, 53075310.
  • Ruppert, M., Ma, X. and Stöckigt, J. (2005) Alkaloid biosynthesis in Rauvolfia– cDNA cloning of major enzymes of the ajmaline pathway. Curr. Org. Chem. 9, 14311444.
  • Ruppert, M., Panjikar, S., Barleben, L. and Stöckigt, J. (2006) Heterologous expression, purification, crystallization and preliminary X-ray analysis of raucaffricine glucosidase, a plant enzyme specifically involved in Rauvolfia alkaloid biosynthesis. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 62, 257260.
  • Samanani, N. and Facchini, P.J. (2002) Purification and characterization of norcoclaurine synthase. The first committed enzyme in benzylisoquinoline alkaloid biosynthesis in plants. J. Biol. Chem. 277, 3387833883.
  • Samanani, N., Liscombe, D.K. and Facchini, P.J. (2004) Molecular cloning and characterization of norcoclaurine synthase, an enzyme catalyzing the first committed step in benzylisoquinoline alkaloid biosynthesis. Plant J. 40, 302313.
  • Samanani, N., Park, S.U. and Facchini, P.J. (2005) Cell type-specific localization of transcripts encoding nine consecutive enzymes involved in protoberberine alkaloid biosynthesis. Plant Cell, 17, 915926.
  • Samanani, N., Alcantara, J., Bourgault, R., Zulak, K.G. and Facchini, P.J. (2006) The role of phloem sieve elements and laticifers in the biosynthesis and accumulation of alkaloids in opium poppy. Plant J. 47, 547563.
  • Sato, F., Inui, T. and Takemura, T. (2007) Metabolic engineering in isoquinoline alkaloid biosynthesis. Curr. Pharm. Biotechnol. 8, 211218.
  • Schröder, G., Unterbusch, E., Kaltenbach, M., Schmidt, J., Strack, D., De Luca, V. and Schröder, J. (1999) Light-induced cytochrome P450-dependent enzyme in indole alkaloid biosynthesis: tabersonine-16-hydroxylase. FEBS Lett. 458, 97102.
  • Schröder, G., Wehinger, E., Lukačin, R., Wellmann, F., Seefelder, W., Schwab, W. and Schröder, J. (2004) Flavonoid methylation: a novel 4′-O-methyltransferase from Catharanthus roseus, and evidence that partially methylated flavanones are substrates of four different flavonoid dioxygenases. Phytochemistry, 65, 10851094.
  • Shanks, J.V., Bhadra, R., Morgan, J., Rijhwani, S. and Vani, S. (1998) Quantification of metabolites in the indole alkaloid pathways of Catharanthus roseus: implications for metabolic engineering. Biotechnol. Bioeng. 58, 333338.
  • Shukla, A.K., Shasany, A.K., Gupta, M.M. and Khanuja, S.P.S. (2006) Transcriptome analysis in Catharanthus roseus leaves and roots for comparative terpenoid indole alkaloid profiles. J. Exp. Bot. 57, 39213932.
  • Sibéril, Y., Benhamron, S., Memelink, J., Giglioli-Guivarc’h, N., Thiersault, M., Boisson, B., Doireau, P. and Gantet, P. (2001) Catharanthus roseus G-box binding factors 1 and 2 act as repressors of strictosidine synthase gene expression in cell cultures. Plant Mol. Biol. 45, 477488.
  • Sottomayor, M., Lopez-Serrano, M., DiCosmo, F. and Ros Barcelo, A. (1998) Purification and characterization of alpha-3′,4′-anhydrovinblastine synthase (peroxidase-like) from Catharanthus roseus (L.) G. Don. FEBS Lett. 428, 299303.
  • Steffens, P., Nagakura, N. and Zenk, M.H. (1985) Purification and characterization of the berberine bridge enzyme from Berberis beaniana cell cultures. Phytochemistry, 24, 25772583.
  • Stevens, L.H., Blom, T.J.M. and Verpoorte, R. (1993) Subcellular localization of tryptophan decarboxylase, strictosidine synthase and strictosidine glucosidase in suspension-cultured cells of Catharanthus roseus and Tabernaemontana divaricata. Plant Cell Rep. 12, 573576.
  • Stöckigt, J., Panjikar, S., Ruppert, M., Barleben, L., Ma, X., Loris, E. and Hill, M. (2007) The molecular architecture of major enzymes from ajmaline biosynthetic pathway. Phytochem. Rev. 6, 1534.
  • St-Pierre, B. and De Luca, V. (1995) A cytochrome P-450 monooxygenase catalyzes the first step in the conversion of tabersonine to vindoline in Catharanthus roseus. Plant Physiol. 109, 131139.
  • St-Pierre, B., Laflamme, P., Alarco, A.M. and De Luca, V. (1998) The terminal O-acetyltransferase involved in vindoline biosynthesis defines a new class of proteins responsible for coenzyme A-dependent acyl transfer. Plant J. 14, 703713.
  • St-Pierre, B., Vazquez-Flota, F.A. and De Luca, V. (1999) Multicellular compartmentation of Catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediate. Plant Cell, 11, 887900.
  • Tabata, M. (1996) The mechanism of shikonin biosynthesis in Lithospermum cell cultures. Plant Tissue Cult. Lett. 13, 117125.
  • Tabata, H. (2004) Paclitaxel production by plant-cell-culture technology. Adv. Biochem. Eng. Biotechnol. 7, 123.
  • Takeshita, N., Fujiwara, H., Mimura, H., Fitchen, J.H., Yamada, Y. and Sato, F. (1995) Molecular cloning and characterization of S-adenosyl-l-methionine:scoulerine-9-O-methyltransferase from cultured cells of Coptis japonica. Plant Cell Physiol. 36, 2936.
  • Tanahashi, T. and Zenk, M.H. (1990) Elicitor induction and characterization of microsomal protopine-6-hydroxylase, the central enzyme in benzophenanthridine alkaloid biosynthesis. Phytochemistry, 29, 11131122.
  • Unterlinner, B., Lenz, R. and Kutchan, T.M. (1999) Molecular cloning and functional expression of codeinone reductase: the penultimate enzyme in morphine biosynthesis in the opium poppy Papaver somniferum. Plant J. 18, 465475.
  • Usia, T., Watabe, T., Kadota, S. and Tezuka, Y. (2005) Cytochrome P450 2D6 (CYP2D6) inhibitory constituents of Catharanthus roseus. Biol. Pharm. Bull. 28, 10211024.
  • Vazquez-Flota, F., De Carolis, E., Alarco, A.M. and De Luca, V. (1997) Molecular cloning and characterization of desacetoxyvindoline-4-hydroxylase, a 2-oxoglutarate dependent-dioxygenase involved in the biosynthesis of vindoline in Catharanthus roseus (L.) G. Don. Plant Mol. Biol. 34, 935948.
  • Veau, B., Courtois, M., Oudin, A., Chénieux, J.C., Rideau, M. and Clastre, M. (2000) Cloning and expression of cDNAs encoding two enzymes of the MEP pathway in Catharanthus roseus. Biochim. Biophys. Acta, 1517, 159163.
  • Viehweger, K., Schwartze, W., Schumann, B., Lein, W. and Roos, W. (2006) The Gα protein controls a pH-dependent signal path to the induction of phytoalexin biosynthesis in Eschscholzia californica. Plant Cell, 18, 15101523.
  • Vom Endt, D., Kijne, J.W. and Memelink, J. (2002) Transcription factors controlling plant secondary metabolism: what regulates the regulators? Phytochemistry, 61, 107114.
  • Vom Endt, D., Soares e Silva, M., Kijne, J.W., Pasquali, G. and Memelink, J. (2007) Identification of a bipartite jasmonate-responsive promoter element in the Catharanthus roseus ORCA3 transcription factor gene that interacts specifically with AT-Hook DNA-binding proteins. Plant Physiol. 144, 16801689.
  • Walz, C., Giavalisco, P., Schad, M., Juenger, M., Klose, J. and Kehr, J. (2004) Proteomics of curcurbit phloem exudate reveals a network of defence proteins. Phytochemistry, 65, 17951804.
  • Weid, M., Ziegler, J. and Kutchan, T.M. (2004) The roles of latex and the vascular bundle in morphine biosynthesis in the opium poppy, Papaver somniferum. Proc. Natl Acad. Sci. USA, 101, 1395713962.
  • Weiss, D., Baumert, A., Vogel, M. and Roos, W. (2006) Sanguinarine reductase, a key enzyme of benzophenanthridine detoxification. Plant Cell Environ. 29, 291302.
  • Whitmer, S., Canel, C., Hallard, D., Goncalves, C. and Verpoorte, R. (1998) Influence of precursor availability on alkaloid accumulation by a transgenic cell line of Catharanthus roseus. Plant Physiol. 116, 853857.
  • Winkel, B.S. (2004) Metabolic channeling in plants. Annu. Rev. Plant Biol. 55, 85107.
  • Winkler, A., Hartner, F., Kutchan, T.M., Glieder, A. and Macheroux, P. (2006) Biochemical evidence that berberine bridge enzyme belongs to a novel family of flavoproteins containing a bi-covalently attached FAD cofactor. J. Biol. Chem. 281, 2127621285.
  • Winkler, A., Kutchan, T.M. and Macheroux, P. (2007) 6-S-cysteinylation of bi-covalently attached FAD in berberine bridge enzyme tunes the redox potential for optimal activity. J. Biol. Chem. 282, 2443724443.
  • Yamamoto, H., Katano, N., Ooi, A. and Inoue, K. (1998) Transformation of loganin and 7-deoxyloganin into secologanin by Lonicera japonica cell suspension cultures. Phytochemistry, 50, 417422.
  • Yamamoto, H., Katano, N., Ooi, A. and Inoue, K. (2000) Secologanin synthase, which catalyzes the oxidative cleavage of loganin into secologanin, is a cytochrome P450. Phytochemistry, 53, 712.
  • Yamazaki, Y., Sudo, H., Yamazaki, M., Aimi, N. and Saito, K. (2003) Camptothecin biosynthetic genes in hairy roots of Ophiorrhiza pumila: cloning, characterization and differential expression in tissues and by stress compounds. Plant Cell Physiol. 44, 395403.
  • Yamazaki, Y., Kitajima, M., Arita, M., Takayama, H., Sudo, H., Yamazaki, M., Aimi, N. and Saito, K. (2004) Biosynthesis of camptothecin. In silico and in vivo tracer study from [1-13C] glucose. Plant Physiol. 134, 161170.
  • Zárate, R. and Verpoorte, R. (2007) Strategies for the genetic modification of the medicinal plant Catharanthus roseus (L.) G. Don. Phytochem. Rev. 6, 475491.
  • Zhao, J. and Verpoorte, R. (2007) Manipulating indole alkaloid production by Catharanthus roseus cell cultures in bioreactors: from biochemical processing to metabolic engineering. Phytochem. Rev. 6, 435457.
  • Ziegler, J., Diaz-Chávez, M.L., Kramell, R., Ammer, C. and Kutchan, T.M. (2005) Comparative macroarray analysis of morphine containing Papaver somniferum and eight morphine free Papaver species identifies an O-methyltransferase involved in benzylisoquinoline biosynthesis. Planta, 222, 458471.
  • Ziegler, J., Voigtländer, S., Schmidt, J., Kramell, R., Miersch, O., Ammer, C., Gesell, A. and Kutchan, T.M. (2006) Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis. Plant J. 48, 177192.
  • Zulak, K.G., Cornish, A., Daskalchuk, T.E., Deyholos, M.K., Goodenowe, D.B., Gordon, P.M.K., Klassen, D., Pelcher, L.E., Sensen, C.W. and Facchini, P.J. (2007) Gene transcript and metabolite profiling of elicitor-induced opium poppy cell cultures reveals the coordinate regulation of primary and secondary metabolism. Planta, 225, 10851106.