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ScienceDirect Making iridoids/secoiridoids and monoterpenoid indole alkaloids: progress on pathway elucidation Vincenzo De Luca1, Vonny Salim1,2, Antje Thamm 1, Sayaka Atsumi Masada1,3 and Fang Yu1,4 Members of the Acanthaceae, Apocynaceae, Bignoniaceae, Caprifoliaceae, Gentianaceae, Labiatae, Lamiaceae, Loasaceae, Loganiaceae, Oleaceae, Plantaginaceae, Rubiaceae, Saxifragaceae, Scrophulariaceae, Valerianaceae, and Verbenaceae plant families are well known to accumulate thousands of bioactive iridoids/secoiridoids while the Apocynaceae, Loganiaceae and Rubiaceae families also accumulate thousands of bioactive monoterpenoid indole alkaloids (MIAs), mostly derived from the secologanin and tryptamine precursors. Several large-scale RNA-sequencing projects have greatly advanced the tools available for identifying candidate genes whose gene products are involved in the biosynthesis of iridoids/MIAs. This has led to the rapid comparative bioinformatics guided elucidation of several key remaining steps in secologanin biosynthesis as well as other steps in MIA biosynthesis. The availability of these tools will permit broad scale biochemical and molecular description of the reactions required for making thousands of iridoid/MIAs. This information will advance our understanding of the evolutionary and ecological roles played by these metabolites in Nature and the genes will be used for biotechnological production of useful iridoids/MIAs. Addresses 1 Department of Biological Sciences, Brock University, 500 Glenridge Avenue, St. Catharines, Ontario L2S 3A1, Canada 2 Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, United States 3 Division of Pharmacognosy, Phytochemistry and Narcotics, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 1588501, Japan 4 School of Biological Engineering, Dalian Polytechnic University, #1 Qinggongyuan, Dalian, Liaoning 116034, China Corresponding authors: De Luca, Vincenzo ([email protected]), Salim, Vonny ([email protected]), Thamm, Antje ([email protected]), Masada, Sayaka Atsumi ([email protected]) and Yu, Fang ([email protected]) Current Opinion in Plant Biology 2014, 19:35–42 This review comes from a themed issue on Physiology and metabolism Edited by Sarah E O’Connor and Thomas P Brutnell
1369-5266/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pbi.2014.03.006
Introduction Plants are the source of many thousands of complex chemicals that include specialized metabolites such as www.sciencedirect.com
phenols, terpenes and alkaloids. The alkaloids alone contain nitrogen in their basic structures that are derived mainly from the a few amino acid precursors and that confer unique biological activities to this class of molecules. In spite of their important pharmacological activities and medicinal uses, the pathways of alkaloid biosynthesis remain poorly understood. This lack of progress has been attributed to the unavailability of the substrate intermediates required for detecting the biochemical reactions in multistep pathways and to inherent difficulties associated with the slow traditional forward genetic approaches for identifying the genes involved. The present review will focus on the recent use of large-scale transcriptome projects [Phytometasyn (http://www.phytometasyn.com/; [1,2]); Medicinal Plant Genomics Consortium (http://www.medicinal plantgenomics.msu.edu/; [2,3,4]); Medicinal Plant Transcriptome Project (http://www.uic.edu/pharmacy/ MedPlTranscriptome/index.html)] and CATHACyc (http://www.cathacyc.org/; [5]) that are speeding up the discovery of monoterpenoid indole alkaloid biosynthesis pathways.
Monoterpenoid indole alkaloids The monoterpenoid indole alkaloids (MIAs) represent one of the largest and most biologically active classes of special metabolites found in thousands of species of plants of the Apocynaceae, Loganiaceae and Rubiaceae families. These MIAs include the Catharanthus roseus vinblastine/vincristine and Camptotheca acuminata/Ophiorhiza pumila camptothecin anticancer alkaloids, the antimalarial quinine from Cinchona ledgeriana/C. succirubra and the antihypertensive drug ajmalicine from Catharanthus rosues/Rauvolfia serpentina. The pathways involved require the condensation of secologanin with tryptamine to yield strictosidine that is then converted to several thousand MIAs found in Nature. While almost 200 MIAs have been described in C. roseus, catharanthine and vindoline are present in greatest abundance and have great commercial value since dimers of these two compounds are drugs used in cancer chemotherapy. The Catharanthus literature related to MIA biosynthesis, its organization in different leaf cell types [6–8] and its biotechnological applications [9] has recently (been) reviewed and has been extensively reviewed over the past 10 years. Current Opinion in Plant Biology 2014, 19:35–42
36 Physiology and metabolism
Missing genes for the supply of geraniol in iridoid biosynthesis Higher plants have cytosolic mevalonic acid and plastidic methyl erythritol phosphate (MEP) pathways that provide isoprenoid precursors for a large range of monoterpene, sesquiterpene, diterpene, triterpene, tetraterpene and polyterpene. In ‘‘range of monoterpene, sesquiterpene, diterpene, triterpene, tetraterpene and polyterpene products’’ roseus, most of MEP pathway genes have been characterized and were shown to be preferentially expressed in specialized Internal Phloem Associated Parenchyma (IPAP) cells (Figure 1; [6,7]) in order to supply precursors for the assembly of iridoid precursors required for the biosynthesis of secologanin in C. roseus. These IPAP cells also preferentially express geraniol 10-hydroxylase (G10H) (Figure 1, reaction 2) and 10-hydroxygeraniol oxidoreductase (10HGO) (Figure 1, reaction 3), while the two terminal reactions in secologanin biosynthesis are known to be preferentially expressed in the leaf epidermis (Figure 1, reactions 8 and 9 [6]). However several genes including isopenenyl diphosphate synthase, geraniol synthase (Figure 1, reaction 1) and other gene products responsible the formation of loganic acid remained to be described (Figure 1, reactions 4–7; iridoid synthase/monoterpene cyclase, 7-deoxyloganetic acid synthase, 7-deoxyloganetic acid glucosyltransferase, and 7-deoxyloganic acid hydroxylase). Isopentenyl diphosphate isomerase (IDI) mediates the equilibrium and supply of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) required for the assembly of geranyl pyrophosphate (GPP). Recent studies have described a single gene in Catharanthus with alternative transcription sites to generate different isoforms of CrIDI that appear to be targeted to peroxisomes, mitochondria and plastids [10]. Presumably the plastid targeted isoform would also be expressed in IPAP cells where it would participate in providing IPP and DMAPP for biosynthesis of GPP. While the MEP pathway is responsible for the formation of IPP and DMAPP, geranyl pyrophosphate synthase (GPPS) is required for the formation of GPP. Recently several CrGPPS genes identified from C. roseus, through database searches (www.ncbi.nlm.nih.gov/and http:// medicinalplantgenomics.msu.edu) [11] were functionally characterized. Three separate types of CrGPPS were functionally characterized with two of them being expressed in chloroplasts and one of them more likely to be targeted to mitochondria. While the results complemented previous studies on the functional characterization and plastid localization of one of these genes [12] several other CrGPPS genes were also described to be present in Catharanthus [11] that make it difficult to establish how many of these genes provide precursor GPP for the biosynthesis of iridoids. Therefore, it is interesting and perhaps understandable that neither Current Opinion in Plant Biology 2014, 19:35–42
study reported if any of these CrGPPS genes might be preferentially expressed in IPAP cells. The molecular cloning of C. roseus geraniol synthase (CrGS) was also recently described and recombinant protein was shown to convert geraniol pyrophosphate (GPP) to geraniol when expressed in Escherichia coli [13]. A significant finding of this study was that CrGS appears to be preferentially expressed in IPAP cells where it converts MEP-derived GPP to produce the geraniol required for the G10H that produces 10-hydroxygeraniol. IPAP cells are known to convert geraniol to 10-oxogeranial via G10H (Figure 1, reaction 2) and 10HGO (Figure 1, reaction 3). Together these characterizations of CrGPPS and CrGS complete the metabolic connection of the MEP pathway to G10H.
Assembly of secologanin Over 2500–3000 iridoids/secoiridoids with a wide variety of biological properties [14,15] have been characterized after isolation from members of the Acanthaceae, Apocynaceae, Bignoniaceae, Caprifoliaceae, Gentianaceae, Labiatae, Lamiaceae, Loasaceae, Loganiaceae, Oleaceae, Plantaginaceae, Rubiaceae, Saxifragaceae, Scrophulariaceae, Valerianaceae, and Verbenaceae plant families. These unusual monoterpenoids contain a methylcyclopentan[c]-pyran skeleton typically fused to a six-membered oxygen containing heterocycle. While iridoids participate in MIA biosynthesis within the Apocynaceae, Loganiaceae and Rubiaceae families this mostly involves a single iridoid, secologanin that provides a C10 or C9 or ocassonally a C8 fragment to the formation of MIAs. However the levels of MIAs produced may be small compared to the levels of iridoids that accumulate within MIA producing plant species. Recent studies in C. roseus have shown that the levels of secologanin in Catharanthus leaves were 10–15 times higher than those of the major MIAs, catharanthine and vindoline [16–18]. This raises important questions about the biological importance of maintaining high levels of secologanin in Catharanthus and perhaps in other MIA accumulating plant species where little documentation exists about the levels of these iridoids. The mobility of iridoids was recently documented when photosynthesis was measured in Snapdragon (Antirrhinum majus) leaves by feeding them with 14CO2 [19]. Measurements showed that 47% of the phloem mobile 14C-photoassimilate was the iridoid antirrhinoside with sucrose making up the rest. The study suggested that rapid conversion of the products of photosynthesis to toxic antirrhinoside could provide a selective advantage to iridoid producing species by deterring herbivory by phloem feeding insects and this was supported in other studies with Alonsoa meridionalis and Asarina barclaiana describing the phloem mobility of antirrhinoside [20]. This raises interesting questions about the mechanisms of iridoid biosynthesis that might involve biochemical cellular specialization events that may be common to iridoid and/or MIA producing plant species www.sciencedirect.com
Iridoid/secoiridoid and monoterpenoid indole alkaloid pathway elucidation De Luca et al. 37
Figure 1
N N H
upper cuticle
CO2CH3
catharanthine
upper epidermis idioblast
laticifer
lower epidermis lower cuticle
H3CO
secologanin tryptamine (9) (11) loganin tryptophan (8) loganic acid
N H
CO2CH3
16-methoxytabersonine
?
?
IPAP
EPAP idioblast
upper & lower epidermis CrTPT2 catharanthine transporter strictosidine (12)
loganic acid (7) 7-deoxyloganic acid (6) 7-deoxyloganetic acid (5) Iridodial (4) 10-oxogeranial (3) 10-hydroxygeraniol (2) geraniol IPAP (1) cells MEP pathway
Laticifers & ldioblasts
H3CO
N CH3
OAc CO2CH3
Last 4 steps in vindoline biosynthesis
Current Opinion in Plant Biology
Biosynthesis of secologanin/MIAs and transporters required for their assembly. The methylerythritol phosphate pathway supplies precursors through geraniol synthase (1) to form loganic acid via geraniol hydroxylase (2), 10-hydroxygeraniol oxidoreductase (3), iridodial cyclase (4), 7-deoxyloganetic acid synthase (5), 7-deoxyloganetic acid glucosyltransferase (6) and deoxyloganic acid 7-hydroxylase (7) in internal phloem associated (IPAP) cells but not in external phloem associated (EPAP) cells. Loganic acid is transported by unknown mechanisms to the leaf epidermis to be converted to secologanin by loganic acid methyltransferase (8) and secologanin synthase (9). The leaf epidermis tryptophan decarboxylase (11) converts tryptophan into tryptamine that is used with secologanin by strictosidine synthase to generate strictosidine. Strictosidine b glucosidase activity generates the labile strictosidine aglycone that serves to produce different ring re-arrangements leading to the formation of corynanthe (example ajmalicine), iboga (example catharanthine) and aspidosperma (example tabersonine) alkaloids through a series of reactions that have not been characterized at the molecular level. The catharanthine is exported through the CrTPT2 transporter to accumulate in the upper and lower cuticles of Catharanthus leaves. Tabersonine is converted by tabersonine-16-hydroxylase and 16-hydroxytabersonine-16-O-methyltransferase to generate 16methoxytabersonine. This MIA is then mobilized by an unknown transport mechanism to laticifers and idioblasts where the remaining four steps in vindoline biosynthesis will take place and vindoline may be transferred to the tonoplast by a vacuolar membrane-associated proton antiport system that remains to be characterized at the molecular level.
such as specialized iridoid biosynthesis IPAP cells (Figure 1). The key cyclization step for conversion of 10-oxogeranial to cis–trans-nepetalactol was identified by using a transcriptomic approach (http://www.medicinalplantgenomics.msu.edu/) to identify NADPH-using enzymes that appeared to be co-regulated with other MIA pathway genes (Figure 1; reaction 4) [21]. While this iridoid cylcase showed 67% identity to progesterone-5breductase that reduces a double bond in the biosynthesis of cardenolides in Digitalis purpurea its expression was www.sciencedirect.com
restricted to the same IPAP cells where G10H is preferentially expressed. While the recombinant iridoid cyclase converted 10-oxogeranial to cis–trans-nepetalactol, it was unable to catalyze the reduction of progesterone. In contrast to all known monoterpene cyclases that involve a cation indermediate produced from geranyl diphosphate, this unique cyclase used 10-oxogeranial in an NAD(P)H requiring reaction. The role of iridoid cyclase was corroborated by virus induced gene silencing (VIGS) since silenced plants expressed lower cyclase levels and accumulated lower levels of the major MIAs, catharanthine and vindoline. Current Opinion in Plant Biology 2014, 19:35–42
38 Physiology and metabolism
The successful identification, cloning and functional characterization of 7-deoxyloganetic acid synthase (7DLS) [16] and 7-deoxyloganic acid 7-hydroxylase (DL7H) [17] involved a bioinformatic search [6,22,23] for homologous cytochrome P 450 (CYP) candidate genes from annotated express sequence tag databases of seven secologanin/MIA producing plant species from the Apocynaceae family, one secologanin/quinoline alkaloid producing species from the Rubiaceae family and one secologanin producing species from the Caprifoliaceae family (Figure 2, databases found at www.phytometasyn.ca). This screen identified three C. roseus candidate genes that were expressed only in secologanin accumulating plant species, but not in dozens of other transcriptome databases from other species of plants that do not accumulate this metabolite. Selected genes were then silenced by VIGS in C. roseus and silenced plants were monitored for a
decline in secologanin/MIA levels (Figure 2; [16,17]). The silencing of 7DLS and DL7H genes triggered significant declines of secologanin/MIAs in suppressed plants and in the case of DL7H suppressed lines they accumulated significant amounts of the DL7H substrate, 7-deoxyloganic acid. Functional expression of the 7DLS and DL7H genes in yeast identified them as authentic CYPs involved in the biosynthesis of 7-deoxyloganetic acid and loganic acid, respectively (Figure 1, reactions 5 and 7)). Gardenia jasminoides that accumulates the iridoids geniposide and gardenoside was used to identify the first known iridoid glucosyltransferase gene [24] from cell cultures that were shown to glucosylate genipin to form geniposide and gardenoside, respectively. The molecular cloning approach involved the design of primers based on a conserved plant secondary product glycosyltransferase
100% 50% 35%
DXS DXR IDI1 MECS GES G10H IS 7DLS UGT7 UGT8 DL7H LAMT SLS TDC STR SGD T16H2 16OMT NMT D4H DAT TPT2
Figure 2
Catharanthus roseus Catharanthus ovalis Catharanthus longifolius Amsonia hubrichtii Vinca minor Rauvolfia serpentina Tabernaemontata elegans Lonicera japonica Cinchona ledgeriana Camptotheca acuminata
Selection of
candidate genes
Toolkit: – gene expression profiling – virus induced gene silencing – targeted metabolite profiling – functional identification of recombinant proteins
rapid pathway discovery Current Opinion in Plant Biology
Candidate gene discovery of biosynthetic pathways through comparative transcriptomics. Comparative heat map of some known MEP, iridoid and MIA pathway enzymes in C. roseus to candidate gene products found in Catharanthus ovalis, Catharanthus longifolius, Amsonia hubrichtii, Vinca minor, Rauvolfia serpentina, Tabernaemontana elegans, Lonicera japonica, Cinchona ledgeriana and Camptotheca acuminata. protein amino. The amino acid sequence similarities range from 33 to100% (Supplementary Table 1). The amino acid identities of genes from different species were imported into Cluster 3.0 (Michiel de Hoon, University of Tokyo; http://bonsai.hgc.jp/mdehoon/software/cluster/) and hierarchical clustering of species was done with uncentered correlation and centroid linkage. The heat map was visualized in Tree View [34]. The selected candidate gene is then tested with a toolkit that might be used in a series of steps available for a plant species, leading to functional gene discovery. Abbreviations of genes in the figure are: [DXS (1-deoxy-D-xylulose-5-phosphate-synthase); DXR (1-deoxy-D-xylulose-5-phosphate reductase); IDII; MECS; (4-cytidyl-diphospho-2-Cmethyl-D-erythritol synthase); GES (geraniol synthase); G10H (geraniol 10-hydroxylase); IS (iridodial cyclase); 7DLS (7-deoxyloganetic acid synthase); UGT7, (7-deoxyloganetic acid glucosyltransferase) UGT8, (weak 7-deoxyloganetic acid glucosyltransferase); DL7H (deoxyloganic acid 7-hydroxylase); LAMT (loganic acid methyltransferase); SLS (secologanin synthase); TDC (tryptophan decarboxylase); STR (strictosidine synthase); SGD (strictosidine b glucosidase); T16H (tabersonine-16-hydroxylase); 16OMT (16-hydroxytabersonine-16-O-methyltransferase); NMT (16-methoxytabersonine-Nmethyltransferase); D4H (deacetoxyvindoline 4-hydroxylase); DAT (deacetylvindoline 4-O-acetyltransferase); TPT2 (catharanthine transporter)]. Current Opinion in Plant Biology 2014, 19:35–42
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Iridoid/secoiridoid and monoterpenoid indole alkaloid pathway elucidation De Luca et al. 39
box that ultimately led to the cloning, functional expression of 13 candidate GTs and the identification of a single candidate that preferentially glucosylated 7-deoxyloganetin and genipin, showed weak activity towards loganetin and was inactive with 7-deoxyloganetic acid. These results strongly suggested that methylation of the carboxyl group occurred before glucosylation in contrast to the results obtained for the loganic acid O-methyltransferase reaction from C. roseus that takes place after glucosyltation [25]. The same molecular approach to clone iridoid GTs in Catharanthus cell cultures combined with mining of a Catharanthus database found in the PlantGDB server (http://plantgdb.org/cgi-bin/blast/ PlantGDBblast) with the Gardenia clone led to the identification of three candidate iridoid GTs [18]. One was a higly specific 7-deoxyloganetic acid GT (UGT8) whose expression was restricted to Catharanthus leaves and was found in a Catharanthus leaf EST database (http://www.phytometasyn.ca/). The other two were 7deoxyloganetin GTs (UGT6 and UGT7) but UGT7 had weak iridoid GT activity and did also glucosylate substrates like curcumin, genistein, luteolin, and kaempferol. In additional studies, tissues were abraded with carborundum particles to preferentially extract RNA from Catharanthus leaf tissues to show that both UGT6 and UGT8 were preferentially expressed within the leaf rather than in the epidermis where the last two steps in secologanin biosynthesis were preferentially expressed. In situ hybridization studies localized expression UGT8 to IPAP cells. The presence of separate gene products (UDP6 and UGT8) that can glucosylate 7-deoxyloganetic acid in leaves and 7-deoxyloganetin in cell suspension cultures, respectively, raise the possibility the separate pathways may be involved in secologanin biosynthesis in leaves and roots. These data as well as other research (reviewed in [26]) indicates that the organization of MIA biosynthesis in roots is quite different from that of leaves. The characterization of root MIA pathways remains an important and key objective that requires urgent attention. These recent discoveries complete the characterization of the eight genes required to form secologanin from geraniol and should facilitate metabolic engineering efforts to produce iridoids or MIAs in plants or in heterologous systems such as yeast [6–8,9]. However it remains to be determined what transport mechanisms might be involved in the export of loganic acid from IPAP cells, its transport to leaf epidermis and its import into these cells for subsequent O-methylation and ring opening to produce secologanin (Figure 1).
Discovery of new MIA pathway genes Several genes involved in MIA biosynthesis have been identified and functionally characterized. These include tryptophan decarboxylase, strictosidine synthase, strictosidine b glucosidase, tabersonine 16-hydroxylase, taberwww.sciencedirect.com
sonine 19-hydroxylase, 16-hydroxytabersonine-16-Omethyltransferase, 2,3-dihydro-3hydroxytabersonine-Ndeacetoxyvindoline-4-hydroxylase methyltransferase, deacetylvindoline-O-acetyltransferase, vacuolar class III peroxidase and NADPH cytochrome C reductase [6– 8,9]. However the genes responsible for numerous reactions that convert strictosidine aglycone and intermediates to form the three key classes of MIAs represent a major challenge to the understanding of their biosynthesis. A recent study showed that T16H is actually encoded by two distinct cytochrome P 450 genes (CYP71D12, CYP71D351) with the newly reported gene (CYP71D351) being the main isoform expressed in Catharanthus leaf epidermis while the second isoform was mainly expressed in floral tissues [27]. The two T16H genes share 82% amino acid sequence identity suggesting a common ancestral origin. While the high substrate specificity of both gene products for tabersonine makes it difficult to understand why independent genes should be expressed in separate Catharanthus organs, this study did help to clarify a number of unexplained results about the complexity of T16H expression in Catharanthus, including the failure to detect the CYP71D12 form of T16H in leaves by in situ hybridization when CYP71D351 form of T16H was successfully detected in leaf epidermis [27]. This raises an important question concerning the cell-type specific expression of the CYP71D12 form of T16H in developing flowers. This information might help to reveal if this form of the enzyme might be involved in other biochemical processes, in addition to this reaction in developing flowers. The organization of MIA biosynthesis in different cell types in C. roseus leaves (Figure 1) show that the last two steps in secologanin biosynthesis, the decarboxylation of tryptamine, the assembly of strictosidine and its conversion to catharanthine and 16-methoxytabersonine occurs in the leaf epidermis while the remaining four steps in vindoline biosynthesis takes place inside the leaf within mesophyll cells and/or in specialized idioblasts/laticifers. The biosynthesis of catharanthine has been suggested to occur in leaf epidermal cells by the almost exclusive presence of this MIA in chloroform extracts of leaf surfaces [28]. In addition, the lack of vindoline in chloroform extracts and its presence within leaves supported the localization of the late stages of vindoline biosynthesis to the mesophyll and provided a plausible explanation for the low levels of vindoline/catharanthine dimers found in Catharanthus leaf tissues. The presence of catharanthine on leaf surfaces and of vindoline within mesophyll was used to suggest that transport mechanisms must be present in order to allow movement of catharanthine and 16-methoxytabersonine from the leaf epidermis to each cellular compartment (Figure 1; [28]). The leaf surface localization of Current Opinion in Plant Biology 2014, 19:35–42
40 Physiology and metabolism
catharanthine stimulated a search for possible alkaloid transporters that might be involved in the secretion process. This led to the discovery [29] of an ATPbinding cassette transporter that is expressed predominantly in the epidermal cells of young leaves and that appears to be responsible for the secretion of catharanthine to the leaf surface [28]. Phylogenetic analysis (http://www.phytometasyn.ca/) suggested that the catharanthine transporter appears to be restricted to plant species active in MIA biosynthesis and is closely related to a common plasma membrane ABC transporter found in Arabidopsis, barley and rice involved in cuticle assembly. These specialized plasma membrane ABC-PDR-catharanthine-like transporters were also identified in plants from distinct geographical origins (Eurasian Vinca minor, African African Tabernamontana elegans, Indian R. serpentina, South American C. ledgeriana, and North American Amsonia hubrichtii). The presence of such a transporter in plants from diverse locations was used to suggest that the appearance of MIA biosynthesis in plants may have occurred coincidentally with the evolution of such secretory mechanisms and that many more plants from this family secrete their MIAs to their respective surfaces [28,29]. A separate recent biochemical study documented the presence in Catharanthus leaves of a vacuolar membrane-associated proton antiport system that requires a pH gradient generated by an ATPase and/or pryrophosphatase tonoplast pump to transport vindoline [30]. This detailed study confirmed earlier research that described such a transporter found in vacuoles isolated from Catharanthus cell cultures [31,32]. Mobilization of vindoline occurred through an active ATP-mediated or PPi-mediated trans-tonoplast transporter that followed Michaelis-Menton saturation kinetics. The study also suggested with less convincing evidence that catharanthine and AVLB might also be transported across tonoplast membranes by the same system. In this context isolated vacuoles were shown to contain similar levels of vindoline and catharanthine, but no AVLB could be detected. It is of great importance that this vindoline transporter be cloned and characterized in order to further elucidate the intercellular organization of MIA biosynthesis (Figure 1).
Future directions This short review highlights the most recent developments in our knowledge of MIA biosynthesis and illustrates how large-scale sequencing projects of non-model medicinal plants is providing novel tools and approaches for identifying target genes by performing mass transcriptome analyses combined with phylogenetic studies in relation to the known phytochemical composition of target plant species. This information combined with tools such as VIGS, carborundum abrasion, in situ hybridization and metabolite profiling are being used to rapidly Current Opinion in Plant Biology 2014, 19:35–42
narrow down candidate genes for functional expression and discovery of their biochemical roles. One of the most important recent developments has been the elucidation of the steps leading to the formation of secologanin. This will have important future impacts for production of iridoids/MIAs in various heterologous microorganisms/ yeast/plants beginning with systems that produce strictosidine, the central precursor of most MIAs, as attested by several personal communications and studies making their way towards publication [33]. These production systems will have a huge impact on the production of any number of iridoids or MIAs that have particular uses in various human activities. In addition the elucidation of the secologanin pathway is likely to have important consequences for the enhanced general discovery of iridoid pathways in any number of desired plant species. Progress will be rapid for elucidating the remaining steps in biosynthesis and transport of vindoline, catharanthine, camptothecin and many other biologically active molecules from plants. The possibility that most MIAs occur on the above ground surfaces of plants needs to be studied and elaborated upon with fundamental studies on the evolutionary processes involved as well as the chemical and environmental ecology that may be responsible selecting this secretory process.
Acknowledgements We wish to express our apologies to colleagues whose work has not been highlighted due to space constraints. We gratefully acknowledge funding from a Discovery Grant provided by National Sciences and Engineering Research Council of Canada and from a Canada Research Chair in Plant Biotechnology to Vincenzo De Luca.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.pbi.2014.03.006.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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Iridoid/secoiridoid and monoterpenoid indole alkaloid pathway elucidation De Luca et al. 41
McKnight TD, O’Connor S, Buell CR: Development of transcriptomic resources for interrogating the biosynthesis of monoterpene indole alkaloids in medicinal plant species. PLoS ONE 2012, 7:e52506. Next generation sequencing and transcriptome assembly of three MIA producing plants (Camptotheca acuminata, Catharanthus roseus, and Rauvolfia serpentina) is reported. Annotations of transcript abundance in different tissues and different treatments is a valuable contribution of this dataset. 4.
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Glenn WS, Runguphan W, O’Connor SE: Recent progress in the metabolic engineering of alkaloids in plant systems. Curr Opin Biotechnol 2013, 24:354-365. Current timely review on recent metabolic engineering efforts to produce MIAs, benzylisoquinoline alkaloids and glucosinolates.
10. Guirimand G, Guihur A, Phillips MA, Oudin A, Gle´varec G, Melin C, Papon N, Clastre M, St-Pierre B, Rodrı´guez-Concepcio´n M et al.: A single gene encodes isopentenyl diphosphate isomerase isoforms targeted to plastids, mitochondria and peroxisomes in Catharanthus roseus. Plant Mol Biol 2012, 79:443-459. 11. Rai AS, Smita SS, Singh AK, Shanker K, Nagegowda DA: Heteromeric and homomeric geranyl diphosphate synthases from Catharanthus roseus and their role in monoterpene indole alkaloid biosynthesis. Mol Plant 2013, 6:1531-1549. 12. Thabet I, Guirimand G, Guihur A, Lanoue A, Courdavault V, Papon N, Bouzid S, Giglioli-Guivarc’h N, Simkin AJ, Clastre M: Characterization and subcellular localization of geranylgeranyl diphosphate synthase from Catharanthus roseus. Mol Biol Rep 2012, 39:3235-3243. 13. Simkin AJ, Miettinen K, Claudel P, Burlat V, Guirimand G, Courdavault V, Papon N, Meyer S, Godet S, St-Pierre B, GiglioliGuivarc’h N, Fischer MJC, Memelink J, Clastre M: Characterization of the plastidial geraniol synthase from Madagascar periwinkle which initiates the monoterpenoid branch of the alkaloid pathway in internal phloem associated parenchyma. Phytochemistry 2013, 85:36-43. 14. Sampaio-Santos MI, Kaplan MAC: Biosynthesis significance of iridoids in chemosystematics. J Braz Chem Soc 2001, 12:144153. 15. Dinda B, Debnath S: Monoterpenes: iridoids. In Natural Products. Edited by Ramawat KG, Me´rillon JM. Berlin/Heidelberg: Springer-Verlag; 2013:3009-3067. 16. Salim V, Wiens B, Masada-Atsumi S, Yu F, De Luca V: 7deoxyloganetic acid synthase catalyzes a key 3 step oxidation to form 7-deoxyloganetic acid in Catharanthus roseus iridoid biosynthesis. Phytochemistry 2014. (in press). www.sciencedirect.com
17. Salim V, Yu F, Altarejos-Caballero J, De Luca V: Virus induced gene silencing identifies Catharanthus roseus 7-deoxyloganic acid 7-hydroxylase, a step in iridoid and monoterpenoid indole alkaloid biosynthesis. Plant J 2013 http://dx.doi.org/10.1111/ tpj.12330. Describe the transcriptomic and associated strategies used to identify and characterize this cytochrome P450. The study highlights how these strategies are facilitating rapid and organized pathway elucidation. 18. Asada K, Salim V, Masada-Atsumi S, Edmunds E, Nagatoshi M, Terasaka K, Mizukami H, De Luca V: Iridoid glucosyltransferase expressed in internal phloem-associated parenchyma cells is involved in the assembly of secologanin and monoterpenoid indole alkaloids in Madagascar periwinkle. Plant Cell 2013, 25:4123-4134. 19. Beninger CW, Cloutier RR, Monteiro MA, Grodzinski B: The distribution of two major iridoids in different organs of Antirrhinum majus L. at selected stages of development. J Chem Ecol 2007, 33:731-747. 20. Voitsekhovskaja OV, Koroleva OA, Batashev DR, Knop C, Tomos AD, Gamalei YV, Heldt HW, Lohaus G: Phloem loading in two scrophulariaceae species. What can drive symplastic flow via plasmodesmata? Plant Physiol 2005, 140:383-395. 21. Geu-Flores F, Sherden NH, Courdavault V, Burlat V, Glenn WS, Wu C, Nims E, Cui Y, O’Connor SE: An alternative route to cyclic terpenes by reductive cyclization in iridoid biosynthesis. Nature 2012, 492:138-142. Reports the key cyclization reaction for biosynthesis of the cyclic terpene precursor to thousands of iridoids from an oxidized geraniol precursor rather than from the universal precursor geranyl diphosphate. 22. De Luca V, Salim V, Atsumi SM, Yu F: Mining the biodiversity of plants: a revolution in the making. Science 2012, 336:16581661. 23. De Luca V, Salim V, Levac D, Atsumi SM, Yu F: Discovery and functional analysis of monoterpenoid indole alkaloid pathways in plants. Methods Enzymol 2012, 515:207-229. 24. Nagatoshi M, Terasaka K, Nagatsu A, Mizukami H: Iridoidspecific glucosyltransferase from Gardenia jasminoides. J Biol Chem 2011, 286:32866-32874. Identifies the first known iridoid glucosyltransferase gene and opens the way to identify glucosyltransferases from other species of iridoid/MIA producing plants. 25. Murata J, Roepke J, Gordon H, De Luca V: The leaf epidermome of Catharanthus roseus reveals its biochemical specialization. Plant Cell 2008, 20:524-542. The study validates the use of carborundum abrasion for harvesting RNA from the epidermis of plant leaves and for producing representative transcriptomes of pathways enriched in this cell type. 26. De Luca V: Monoterpenoid indole alkaloid biosynthesis. In Plant Metabolism and Biotechnology, edn 1. Edited by Ashihara H, Crozier A, Komamine A.2011:263-291. 27. Besseau S, Kellner F, Lanoue A, Thamm AMK, Salim V, Schneider B, Geu-Flores B, Ho¨fer R, Guirimand G, Guihur A, Oudin A, Glevarec G, Foureau E, Papon N, Clastre M, GiglioliGuivarc’h N, St-Pierre B, Werck-Reichhart D, Burlat V, De Luca V, O’Connor SE, Courdavault V: A pair of tabersonine 16hydroxylases initiates the synthesis of vindoline in an organdependent manner in Catharanthus roseus. Plant Physiol 2013, 163:1792-1803. The study shows the importance of harvesting genes from organs actually producing a given metabolite for identifying genes actually involve in its biosynthesis. 28. Roepke J, Salim V, Thamm AJK, Murata J, Ploss K, Boland W, De Luca V: Vinca drug components accumulate exclusively in leaf exudates of Madagascar periwinkle. Proc Natl Acad Sci U S A 2010, 107:15287-15292. The study explains that low levels of anticancer drugs are made in Catharanthus roseus because the plant sequesters the precursors in separate locations. It also suggests the MIA biosynthesis in plants may have evolved with secretory processes that ensure their presence on the above ground plant surfaces where they accomplished unknown biological roles. 29. Yu F, De Luca V: ATP-binding cassette transporter controls leaf surface secretion of anticancer drug components in Current Opinion in Plant Biology 2014, 19:35–42
42 Physiology and metabolism
Catharanthus roseus. Proc Natl Acad Sci U S A 2013, 110:1583015835. The secretion of catharanthine and perhaps other MIAs was shown to be mediated by a plasma membrane associated transporter related to a common transporter involved in cuticle formation. This type of transporter was also identified in other MIA producing plant species and provides support to promote further studies on the secretory nature of MIA as a common feature of MIA accumulating plant species. 30. Carqueijeiro I, Noronha H, Duarte P, Gero´s H, Sottomayor M: Vacuolar transport of the medicinal alkaloids from Catharanthus roseus is mediated by a proton-driven antiport. Plant Physiol 2013, 162:1486-1496. A tonoplast associated proton-driven antiporter is carefully characterized at the biochemical level, providing some important insights for the mobilization and storage of vindoline in Catharanthus leaves. The study explores some controversial aspects of MIA transport in relation to the complexities involved.
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31. Deus-Neumann B, Zenk MH: A highly selective alkaloid uptake system in vacuoles of higher plants. Planta 1984, 162:250-260. 32. Deus-Neumann B, Zenk MH: Accumulation of alkaloids in plant vacuoles does not involve an ion-trap mechanism. Planta 1986, 167:44-53. 33. Ho¨fer R, Dong L, Andre´ F, Ginglinger JF, Lugan R, Gavira C, Grec S, Lang G, Memelink J, Van Der Krol S, Bouwmeester H, Werck-Reichhart D: Geraniol hydroxylase and hydroxygeraniol oxidase activities of the CYP76 family of cytochrome P450 enzymes and potential for engineering the early steps of the (seco)iridoid pathway. Metab Eng 2013 http://dx.doi.org/ 10.1016/j.ymben.2013.08.001i. 34. Saldanha: Java Treeview — extensible visualization of microarray data. Bioinformatics 2004, 20(17):3246-3248.
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ScienceDirect Making iridoids/secoiridoids and monoterpenoid indole alkaloids: progress on pathway elucidation Vincenzo De Luca1, Vonny Salim1,2, Antje Thamm 1, Sayaka Atsumi Masada1,3 and Fang Yu1,4 Members of the Acanthaceae, Apocynaceae, Bignoniaceae, Caprifoliaceae, Gentianaceae, Labiatae, Lamiaceae, Loasaceae, Loganiaceae, Oleaceae, Plantaginaceae, Rubiaceae, Saxifragaceae, Scrophulariaceae, Valerianaceae, and Verbenaceae plant families are well known to accumulate thousands of bioactive iridoids/secoiridoids while the Apocynaceae, Loganiaceae and Rubiaceae families also accumulate thousands of bioactive monoterpenoid indole alkaloids (MIAs), mostly derived from the secologanin and tryptamine precursors. Several large-scale RNA-sequencing projects have greatly advanced the tools available for identifying candidate genes whose gene products are involved in the biosynthesis of iridoids/MIAs. This has led to the rapid comparative bioinformatics guided elucidation of several key remaining steps in secologanin biosynthesis as well as other steps in MIA biosynthesis. The availability of these tools will permit broad scale biochemical and molecular description of the reactions required for making thousands of iridoid/MIAs. This information will advance our understanding of the evolutionary and ecological roles played by these metabolites in Nature and the genes will be used for biotechnological production of useful iridoids/MIAs. Addresses 1 Department of Biological Sciences, Brock University, 500 Glenridge Avenue, St. Catharines, Ontario L2S 3A1, Canada 2 Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, United States 3 Division of Pharmacognosy, Phytochemistry and Narcotics, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 1588501, Japan 4 School of Biological Engineering, Dalian Polytechnic University, #1 Qinggongyuan, Dalian, Liaoning 116034, China Corresponding authors: De Luca, Vincenzo ([email protected]), Salim, Vonny ([email protected]), Thamm, Antje ([email protected]), Masada, Sayaka Atsumi ([email protected]) and Yu, Fang ([email protected]) Current Opinion in Plant Biology 2014, 19:35–42 This review comes from a themed issue on Physiology and metabolism Edited by Sarah E O’Connor and Thomas P Brutnell
1369-5266/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pbi.2014.03.006
Introduction Plants are the source of many thousands of complex chemicals that include specialized metabolites such as www.sciencedirect.com
phenols, terpenes and alkaloids. The alkaloids alone contain nitrogen in their basic structures that are derived mainly from the a few amino acid precursors and that confer unique biological activities to this class of molecules. In spite of their important pharmacological activities and medicinal uses, the pathways of alkaloid biosynthesis remain poorly understood. This lack of progress has been attributed to the unavailability of the substrate intermediates required for detecting the biochemical reactions in multistep pathways and to inherent difficulties associated with the slow traditional forward genetic approaches for identifying the genes involved. The present review will focus on the recent use of large-scale transcriptome projects [Phytometasyn (http://www.phytometasyn.com/; [1,2]); Medicinal Plant Genomics Consortium (http://www.medicinal plantgenomics.msu.edu/; [2,3,4]); Medicinal Plant Transcriptome Project (http://www.uic.edu/pharmacy/ MedPlTranscriptome/index.html)] and CATHACyc (http://www.cathacyc.org/; [5]) that are speeding up the discovery of monoterpenoid indole alkaloid biosynthesis pathways.
Monoterpenoid indole alkaloids The monoterpenoid indole alkaloids (MIAs) represent one of the largest and most biologically active classes of special metabolites found in thousands of species of plants of the Apocynaceae, Loganiaceae and Rubiaceae families. These MIAs include the Catharanthus roseus vinblastine/vincristine and Camptotheca acuminata/Ophiorhiza pumila camptothecin anticancer alkaloids, the antimalarial quinine from Cinchona ledgeriana/C. succirubra and the antihypertensive drug ajmalicine from Catharanthus rosues/Rauvolfia serpentina. The pathways involved require the condensation of secologanin with tryptamine to yield strictosidine that is then converted to several thousand MIAs found in Nature. While almost 200 MIAs have been described in C. roseus, catharanthine and vindoline are present in greatest abundance and have great commercial value since dimers of these two compounds are drugs used in cancer chemotherapy. The Catharanthus literature related to MIA biosynthesis, its organization in different leaf cell types [6–8] and its biotechnological applications [9] has recently (been) reviewed and has been extensively reviewed over the past 10 years. Current Opinion in Plant Biology 2014, 19:35–42
36 Physiology and metabolism
Missing genes for the supply of geraniol in iridoid biosynthesis Higher plants have cytosolic mevalonic acid and plastidic methyl erythritol phosphate (MEP) pathways that provide isoprenoid precursors for a large range of monoterpene, sesquiterpene, diterpene, triterpene, tetraterpene and polyterpene. In ‘‘range of monoterpene, sesquiterpene, diterpene, triterpene, tetraterpene and polyterpene products’’ roseus, most of MEP pathway genes have been characterized and were shown to be preferentially expressed in specialized Internal Phloem Associated Parenchyma (IPAP) cells (Figure 1; [6,7]) in order to supply precursors for the assembly of iridoid precursors required for the biosynthesis of secologanin in C. roseus. These IPAP cells also preferentially express geraniol 10-hydroxylase (G10H) (Figure 1, reaction 2) and 10-hydroxygeraniol oxidoreductase (10HGO) (Figure 1, reaction 3), while the two terminal reactions in secologanin biosynthesis are known to be preferentially expressed in the leaf epidermis (Figure 1, reactions 8 and 9 [6]). However several genes including isopenenyl diphosphate synthase, geraniol synthase (Figure 1, reaction 1) and other gene products responsible the formation of loganic acid remained to be described (Figure 1, reactions 4–7; iridoid synthase/monoterpene cyclase, 7-deoxyloganetic acid synthase, 7-deoxyloganetic acid glucosyltransferase, and 7-deoxyloganic acid hydroxylase). Isopentenyl diphosphate isomerase (IDI) mediates the equilibrium and supply of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) required for the assembly of geranyl pyrophosphate (GPP). Recent studies have described a single gene in Catharanthus with alternative transcription sites to generate different isoforms of CrIDI that appear to be targeted to peroxisomes, mitochondria and plastids [10]. Presumably the plastid targeted isoform would also be expressed in IPAP cells where it would participate in providing IPP and DMAPP for biosynthesis of GPP. While the MEP pathway is responsible for the formation of IPP and DMAPP, geranyl pyrophosphate synthase (GPPS) is required for the formation of GPP. Recently several CrGPPS genes identified from C. roseus, through database searches (www.ncbi.nlm.nih.gov/and http:// medicinalplantgenomics.msu.edu) [11] were functionally characterized. Three separate types of CrGPPS were functionally characterized with two of them being expressed in chloroplasts and one of them more likely to be targeted to mitochondria. While the results complemented previous studies on the functional characterization and plastid localization of one of these genes [12] several other CrGPPS genes were also described to be present in Catharanthus [11] that make it difficult to establish how many of these genes provide precursor GPP for the biosynthesis of iridoids. Therefore, it is interesting and perhaps understandable that neither Current Opinion in Plant Biology 2014, 19:35–42
study reported if any of these CrGPPS genes might be preferentially expressed in IPAP cells. The molecular cloning of C. roseus geraniol synthase (CrGS) was also recently described and recombinant protein was shown to convert geraniol pyrophosphate (GPP) to geraniol when expressed in Escherichia coli [13]. A significant finding of this study was that CrGS appears to be preferentially expressed in IPAP cells where it converts MEP-derived GPP to produce the geraniol required for the G10H that produces 10-hydroxygeraniol. IPAP cells are known to convert geraniol to 10-oxogeranial via G10H (Figure 1, reaction 2) and 10HGO (Figure 1, reaction 3). Together these characterizations of CrGPPS and CrGS complete the metabolic connection of the MEP pathway to G10H.
Assembly of secologanin Over 2500–3000 iridoids/secoiridoids with a wide variety of biological properties [14,15] have been characterized after isolation from members of the Acanthaceae, Apocynaceae, Bignoniaceae, Caprifoliaceae, Gentianaceae, Labiatae, Lamiaceae, Loasaceae, Loganiaceae, Oleaceae, Plantaginaceae, Rubiaceae, Saxifragaceae, Scrophulariaceae, Valerianaceae, and Verbenaceae plant families. These unusual monoterpenoids contain a methylcyclopentan[c]-pyran skeleton typically fused to a six-membered oxygen containing heterocycle. While iridoids participate in MIA biosynthesis within the Apocynaceae, Loganiaceae and Rubiaceae families this mostly involves a single iridoid, secologanin that provides a C10 or C9 or ocassonally a C8 fragment to the formation of MIAs. However the levels of MIAs produced may be small compared to the levels of iridoids that accumulate within MIA producing plant species. Recent studies in C. roseus have shown that the levels of secologanin in Catharanthus leaves were 10–15 times higher than those of the major MIAs, catharanthine and vindoline [16–18]. This raises important questions about the biological importance of maintaining high levels of secologanin in Catharanthus and perhaps in other MIA accumulating plant species where little documentation exists about the levels of these iridoids. The mobility of iridoids was recently documented when photosynthesis was measured in Snapdragon (Antirrhinum majus) leaves by feeding them with 14CO2 [19]. Measurements showed that 47% of the phloem mobile 14C-photoassimilate was the iridoid antirrhinoside with sucrose making up the rest. The study suggested that rapid conversion of the products of photosynthesis to toxic antirrhinoside could provide a selective advantage to iridoid producing species by deterring herbivory by phloem feeding insects and this was supported in other studies with Alonsoa meridionalis and Asarina barclaiana describing the phloem mobility of antirrhinoside [20]. This raises interesting questions about the mechanisms of iridoid biosynthesis that might involve biochemical cellular specialization events that may be common to iridoid and/or MIA producing plant species www.sciencedirect.com
Iridoid/secoiridoid and monoterpenoid indole alkaloid pathway elucidation De Luca et al. 37
Figure 1
N N H
upper cuticle
CO2CH3
catharanthine
upper epidermis idioblast
laticifer
lower epidermis lower cuticle
H3CO
secologanin tryptamine (9) (11) loganin tryptophan (8) loganic acid
N H
CO2CH3
16-methoxytabersonine
?
?
IPAP
EPAP idioblast
upper & lower epidermis CrTPT2 catharanthine transporter strictosidine (12)
loganic acid (7) 7-deoxyloganic acid (6) 7-deoxyloganetic acid (5) Iridodial (4) 10-oxogeranial (3) 10-hydroxygeraniol (2) geraniol IPAP (1) cells MEP pathway
Laticifers & ldioblasts
H3CO
N CH3
OAc CO2CH3
Last 4 steps in vindoline biosynthesis
Current Opinion in Plant Biology
Biosynthesis of secologanin/MIAs and transporters required for their assembly. The methylerythritol phosphate pathway supplies precursors through geraniol synthase (1) to form loganic acid via geraniol hydroxylase (2), 10-hydroxygeraniol oxidoreductase (3), iridodial cyclase (4), 7-deoxyloganetic acid synthase (5), 7-deoxyloganetic acid glucosyltransferase (6) and deoxyloganic acid 7-hydroxylase (7) in internal phloem associated (IPAP) cells but not in external phloem associated (EPAP) cells. Loganic acid is transported by unknown mechanisms to the leaf epidermis to be converted to secologanin by loganic acid methyltransferase (8) and secologanin synthase (9). The leaf epidermis tryptophan decarboxylase (11) converts tryptophan into tryptamine that is used with secologanin by strictosidine synthase to generate strictosidine. Strictosidine b glucosidase activity generates the labile strictosidine aglycone that serves to produce different ring re-arrangements leading to the formation of corynanthe (example ajmalicine), iboga (example catharanthine) and aspidosperma (example tabersonine) alkaloids through a series of reactions that have not been characterized at the molecular level. The catharanthine is exported through the CrTPT2 transporter to accumulate in the upper and lower cuticles of Catharanthus leaves. Tabersonine is converted by tabersonine-16-hydroxylase and 16-hydroxytabersonine-16-O-methyltransferase to generate 16methoxytabersonine. This MIA is then mobilized by an unknown transport mechanism to laticifers and idioblasts where the remaining four steps in vindoline biosynthesis will take place and vindoline may be transferred to the tonoplast by a vacuolar membrane-associated proton antiport system that remains to be characterized at the molecular level.
such as specialized iridoid biosynthesis IPAP cells (Figure 1). The key cyclization step for conversion of 10-oxogeranial to cis–trans-nepetalactol was identified by using a transcriptomic approach (http://www.medicinalplantgenomics.msu.edu/) to identify NADPH-using enzymes that appeared to be co-regulated with other MIA pathway genes (Figure 1; reaction 4) [21]. While this iridoid cylcase showed 67% identity to progesterone-5breductase that reduces a double bond in the biosynthesis of cardenolides in Digitalis purpurea its expression was www.sciencedirect.com
restricted to the same IPAP cells where G10H is preferentially expressed. While the recombinant iridoid cyclase converted 10-oxogeranial to cis–trans-nepetalactol, it was unable to catalyze the reduction of progesterone. In contrast to all known monoterpene cyclases that involve a cation indermediate produced from geranyl diphosphate, this unique cyclase used 10-oxogeranial in an NAD(P)H requiring reaction. The role of iridoid cyclase was corroborated by virus induced gene silencing (VIGS) since silenced plants expressed lower cyclase levels and accumulated lower levels of the major MIAs, catharanthine and vindoline. Current Opinion in Plant Biology 2014, 19:35–42
38 Physiology and metabolism
The successful identification, cloning and functional characterization of 7-deoxyloganetic acid synthase (7DLS) [16] and 7-deoxyloganic acid 7-hydroxylase (DL7H) [17] involved a bioinformatic search [6,22,23] for homologous cytochrome P 450 (CYP) candidate genes from annotated express sequence tag databases of seven secologanin/MIA producing plant species from the Apocynaceae family, one secologanin/quinoline alkaloid producing species from the Rubiaceae family and one secologanin producing species from the Caprifoliaceae family (Figure 2, databases found at www.phytometasyn.ca). This screen identified three C. roseus candidate genes that were expressed only in secologanin accumulating plant species, but not in dozens of other transcriptome databases from other species of plants that do not accumulate this metabolite. Selected genes were then silenced by VIGS in C. roseus and silenced plants were monitored for a
decline in secologanin/MIA levels (Figure 2; [16,17]). The silencing of 7DLS and DL7H genes triggered significant declines of secologanin/MIAs in suppressed plants and in the case of DL7H suppressed lines they accumulated significant amounts of the DL7H substrate, 7-deoxyloganic acid. Functional expression of the 7DLS and DL7H genes in yeast identified them as authentic CYPs involved in the biosynthesis of 7-deoxyloganetic acid and loganic acid, respectively (Figure 1, reactions 5 and 7)). Gardenia jasminoides that accumulates the iridoids geniposide and gardenoside was used to identify the first known iridoid glucosyltransferase gene [24] from cell cultures that were shown to glucosylate genipin to form geniposide and gardenoside, respectively. The molecular cloning approach involved the design of primers based on a conserved plant secondary product glycosyltransferase
100% 50% 35%
DXS DXR IDI1 MECS GES G10H IS 7DLS UGT7 UGT8 DL7H LAMT SLS TDC STR SGD T16H2 16OMT NMT D4H DAT TPT2
Figure 2
Catharanthus roseus Catharanthus ovalis Catharanthus longifolius Amsonia hubrichtii Vinca minor Rauvolfia serpentina Tabernaemontata elegans Lonicera japonica Cinchona ledgeriana Camptotheca acuminata
Selection of
candidate genes
Toolkit: – gene expression profiling – virus induced gene silencing – targeted metabolite profiling – functional identification of recombinant proteins
rapid pathway discovery Current Opinion in Plant Biology
Candidate gene discovery of biosynthetic pathways through comparative transcriptomics. Comparative heat map of some known MEP, iridoid and MIA pathway enzymes in C. roseus to candidate gene products found in Catharanthus ovalis, Catharanthus longifolius, Amsonia hubrichtii, Vinca minor, Rauvolfia serpentina, Tabernaemontana elegans, Lonicera japonica, Cinchona ledgeriana and Camptotheca acuminata. protein amino. The amino acid sequence similarities range from 33 to100% (Supplementary Table 1). The amino acid identities of genes from different species were imported into Cluster 3.0 (Michiel de Hoon, University of Tokyo; http://bonsai.hgc.jp/mdehoon/software/cluster/) and hierarchical clustering of species was done with uncentered correlation and centroid linkage. The heat map was visualized in Tree View [34]. The selected candidate gene is then tested with a toolkit that might be used in a series of steps available for a plant species, leading to functional gene discovery. Abbreviations of genes in the figure are: [DXS (1-deoxy-D-xylulose-5-phosphate-synthase); DXR (1-deoxy-D-xylulose-5-phosphate reductase); IDII; MECS; (4-cytidyl-diphospho-2-Cmethyl-D-erythritol synthase); GES (geraniol synthase); G10H (geraniol 10-hydroxylase); IS (iridodial cyclase); 7DLS (7-deoxyloganetic acid synthase); UGT7, (7-deoxyloganetic acid glucosyltransferase) UGT8, (weak 7-deoxyloganetic acid glucosyltransferase); DL7H (deoxyloganic acid 7-hydroxylase); LAMT (loganic acid methyltransferase); SLS (secologanin synthase); TDC (tryptophan decarboxylase); STR (strictosidine synthase); SGD (strictosidine b glucosidase); T16H (tabersonine-16-hydroxylase); 16OMT (16-hydroxytabersonine-16-O-methyltransferase); NMT (16-methoxytabersonine-Nmethyltransferase); D4H (deacetoxyvindoline 4-hydroxylase); DAT (deacetylvindoline 4-O-acetyltransferase); TPT2 (catharanthine transporter)]. Current Opinion in Plant Biology 2014, 19:35–42
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Iridoid/secoiridoid and monoterpenoid indole alkaloid pathway elucidation De Luca et al. 39
box that ultimately led to the cloning, functional expression of 13 candidate GTs and the identification of a single candidate that preferentially glucosylated 7-deoxyloganetin and genipin, showed weak activity towards loganetin and was inactive with 7-deoxyloganetic acid. These results strongly suggested that methylation of the carboxyl group occurred before glucosylation in contrast to the results obtained for the loganic acid O-methyltransferase reaction from C. roseus that takes place after glucosyltation [25]. The same molecular approach to clone iridoid GTs in Catharanthus cell cultures combined with mining of a Catharanthus database found in the PlantGDB server (http://plantgdb.org/cgi-bin/blast/ PlantGDBblast) with the Gardenia clone led to the identification of three candidate iridoid GTs [18]. One was a higly specific 7-deoxyloganetic acid GT (UGT8) whose expression was restricted to Catharanthus leaves and was found in a Catharanthus leaf EST database (http://www.phytometasyn.ca/). The other two were 7deoxyloganetin GTs (UGT6 and UGT7) but UGT7 had weak iridoid GT activity and did also glucosylate substrates like curcumin, genistein, luteolin, and kaempferol. In additional studies, tissues were abraded with carborundum particles to preferentially extract RNA from Catharanthus leaf tissues to show that both UGT6 and UGT8 were preferentially expressed within the leaf rather than in the epidermis where the last two steps in secologanin biosynthesis were preferentially expressed. In situ hybridization studies localized expression UGT8 to IPAP cells. The presence of separate gene products (UDP6 and UGT8) that can glucosylate 7-deoxyloganetic acid in leaves and 7-deoxyloganetin in cell suspension cultures, respectively, raise the possibility the separate pathways may be involved in secologanin biosynthesis in leaves and roots. These data as well as other research (reviewed in [26]) indicates that the organization of MIA biosynthesis in roots is quite different from that of leaves. The characterization of root MIA pathways remains an important and key objective that requires urgent attention. These recent discoveries complete the characterization of the eight genes required to form secologanin from geraniol and should facilitate metabolic engineering efforts to produce iridoids or MIAs in plants or in heterologous systems such as yeast [6–8,9]. However it remains to be determined what transport mechanisms might be involved in the export of loganic acid from IPAP cells, its transport to leaf epidermis and its import into these cells for subsequent O-methylation and ring opening to produce secologanin (Figure 1).
Discovery of new MIA pathway genes Several genes involved in MIA biosynthesis have been identified and functionally characterized. These include tryptophan decarboxylase, strictosidine synthase, strictosidine b glucosidase, tabersonine 16-hydroxylase, taberwww.sciencedirect.com
sonine 19-hydroxylase, 16-hydroxytabersonine-16-Omethyltransferase, 2,3-dihydro-3hydroxytabersonine-Ndeacetoxyvindoline-4-hydroxylase methyltransferase, deacetylvindoline-O-acetyltransferase, vacuolar class III peroxidase and NADPH cytochrome C reductase [6– 8,9]. However the genes responsible for numerous reactions that convert strictosidine aglycone and intermediates to form the three key classes of MIAs represent a major challenge to the understanding of their biosynthesis. A recent study showed that T16H is actually encoded by two distinct cytochrome P 450 genes (CYP71D12, CYP71D351) with the newly reported gene (CYP71D351) being the main isoform expressed in Catharanthus leaf epidermis while the second isoform was mainly expressed in floral tissues [27]. The two T16H genes share 82% amino acid sequence identity suggesting a common ancestral origin. While the high substrate specificity of both gene products for tabersonine makes it difficult to understand why independent genes should be expressed in separate Catharanthus organs, this study did help to clarify a number of unexplained results about the complexity of T16H expression in Catharanthus, including the failure to detect the CYP71D12 form of T16H in leaves by in situ hybridization when CYP71D351 form of T16H was successfully detected in leaf epidermis [27]. This raises an important question concerning the cell-type specific expression of the CYP71D12 form of T16H in developing flowers. This information might help to reveal if this form of the enzyme might be involved in other biochemical processes, in addition to this reaction in developing flowers. The organization of MIA biosynthesis in different cell types in C. roseus leaves (Figure 1) show that the last two steps in secologanin biosynthesis, the decarboxylation of tryptamine, the assembly of strictosidine and its conversion to catharanthine and 16-methoxytabersonine occurs in the leaf epidermis while the remaining four steps in vindoline biosynthesis takes place inside the leaf within mesophyll cells and/or in specialized idioblasts/laticifers. The biosynthesis of catharanthine has been suggested to occur in leaf epidermal cells by the almost exclusive presence of this MIA in chloroform extracts of leaf surfaces [28]. In addition, the lack of vindoline in chloroform extracts and its presence within leaves supported the localization of the late stages of vindoline biosynthesis to the mesophyll and provided a plausible explanation for the low levels of vindoline/catharanthine dimers found in Catharanthus leaf tissues. The presence of catharanthine on leaf surfaces and of vindoline within mesophyll was used to suggest that transport mechanisms must be present in order to allow movement of catharanthine and 16-methoxytabersonine from the leaf epidermis to each cellular compartment (Figure 1; [28]). The leaf surface localization of Current Opinion in Plant Biology 2014, 19:35–42
40 Physiology and metabolism
catharanthine stimulated a search for possible alkaloid transporters that might be involved in the secretion process. This led to the discovery [29] of an ATPbinding cassette transporter that is expressed predominantly in the epidermal cells of young leaves and that appears to be responsible for the secretion of catharanthine to the leaf surface [28]. Phylogenetic analysis (http://www.phytometasyn.ca/) suggested that the catharanthine transporter appears to be restricted to plant species active in MIA biosynthesis and is closely related to a common plasma membrane ABC transporter found in Arabidopsis, barley and rice involved in cuticle assembly. These specialized plasma membrane ABC-PDR-catharanthine-like transporters were also identified in plants from distinct geographical origins (Eurasian Vinca minor, African African Tabernamontana elegans, Indian R. serpentina, South American C. ledgeriana, and North American Amsonia hubrichtii). The presence of such a transporter in plants from diverse locations was used to suggest that the appearance of MIA biosynthesis in plants may have occurred coincidentally with the evolution of such secretory mechanisms and that many more plants from this family secrete their MIAs to their respective surfaces [28,29]. A separate recent biochemical study documented the presence in Catharanthus leaves of a vacuolar membrane-associated proton antiport system that requires a pH gradient generated by an ATPase and/or pryrophosphatase tonoplast pump to transport vindoline [30]. This detailed study confirmed earlier research that described such a transporter found in vacuoles isolated from Catharanthus cell cultures [31,32]. Mobilization of vindoline occurred through an active ATP-mediated or PPi-mediated trans-tonoplast transporter that followed Michaelis-Menton saturation kinetics. The study also suggested with less convincing evidence that catharanthine and AVLB might also be transported across tonoplast membranes by the same system. In this context isolated vacuoles were shown to contain similar levels of vindoline and catharanthine, but no AVLB could be detected. It is of great importance that this vindoline transporter be cloned and characterized in order to further elucidate the intercellular organization of MIA biosynthesis (Figure 1).
Future directions This short review highlights the most recent developments in our knowledge of MIA biosynthesis and illustrates how large-scale sequencing projects of non-model medicinal plants is providing novel tools and approaches for identifying target genes by performing mass transcriptome analyses combined with phylogenetic studies in relation to the known phytochemical composition of target plant species. This information combined with tools such as VIGS, carborundum abrasion, in situ hybridization and metabolite profiling are being used to rapidly Current Opinion in Plant Biology 2014, 19:35–42
narrow down candidate genes for functional expression and discovery of their biochemical roles. One of the most important recent developments has been the elucidation of the steps leading to the formation of secologanin. This will have important future impacts for production of iridoids/MIAs in various heterologous microorganisms/ yeast/plants beginning with systems that produce strictosidine, the central precursor of most MIAs, as attested by several personal communications and studies making their way towards publication [33]. These production systems will have a huge impact on the production of any number of iridoids or MIAs that have particular uses in various human activities. In addition the elucidation of the secologanin pathway is likely to have important consequences for the enhanced general discovery of iridoid pathways in any number of desired plant species. Progress will be rapid for elucidating the remaining steps in biosynthesis and transport of vindoline, catharanthine, camptothecin and many other biologically active molecules from plants. The possibility that most MIAs occur on the above ground surfaces of plants needs to be studied and elaborated upon with fundamental studies on the evolutionary processes involved as well as the chemical and environmental ecology that may be responsible selecting this secretory process.
Acknowledgements We wish to express our apologies to colleagues whose work has not been highlighted due to space constraints. We gratefully acknowledge funding from a Discovery Grant provided by National Sciences and Engineering Research Council of Canada and from a Canada Research Chair in Plant Biotechnology to Vincenzo De Luca.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.pbi.2014.03.006.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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McKnight TD, O’Connor S, Buell CR: Development of transcriptomic resources for interrogating the biosynthesis of monoterpene indole alkaloids in medicinal plant species. PLoS ONE 2012, 7:e52506. Next generation sequencing and transcriptome assembly of three MIA producing plants (Camptotheca acuminata, Catharanthus roseus, and Rauvolfia serpentina) is reported. Annotations of transcript abundance in different tissues and different treatments is a valuable contribution of this dataset. 4.
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17. Salim V, Yu F, Altarejos-Caballero J, De Luca V: Virus induced gene silencing identifies Catharanthus roseus 7-deoxyloganic acid 7-hydroxylase, a step in iridoid and monoterpenoid indole alkaloid biosynthesis. Plant J 2013 http://dx.doi.org/10.1111/ tpj.12330. Describe the transcriptomic and associated strategies used to identify and characterize this cytochrome P450. The study highlights how these strategies are facilitating rapid and organized pathway elucidation. 18. Asada K, Salim V, Masada-Atsumi S, Edmunds E, Nagatoshi M, Terasaka K, Mizukami H, De Luca V: Iridoid glucosyltransferase expressed in internal phloem-associated parenchyma cells is involved in the assembly of secologanin and monoterpenoid indole alkaloids in Madagascar periwinkle. Plant Cell 2013, 25:4123-4134. 19. Beninger CW, Cloutier RR, Monteiro MA, Grodzinski B: The distribution of two major iridoids in different organs of Antirrhinum majus L. at selected stages of development. J Chem Ecol 2007, 33:731-747. 20. Voitsekhovskaja OV, Koroleva OA, Batashev DR, Knop C, Tomos AD, Gamalei YV, Heldt HW, Lohaus G: Phloem loading in two scrophulariaceae species. What can drive symplastic flow via plasmodesmata? Plant Physiol 2005, 140:383-395. 21. Geu-Flores F, Sherden NH, Courdavault V, Burlat V, Glenn WS, Wu C, Nims E, Cui Y, O’Connor SE: An alternative route to cyclic terpenes by reductive cyclization in iridoid biosynthesis. Nature 2012, 492:138-142. Reports the key cyclization reaction for biosynthesis of the cyclic terpene precursor to thousands of iridoids from an oxidized geraniol precursor rather than from the universal precursor geranyl diphosphate. 22. De Luca V, Salim V, Atsumi SM, Yu F: Mining the biodiversity of plants: a revolution in the making. Science 2012, 336:16581661. 23. De Luca V, Salim V, Levac D, Atsumi SM, Yu F: Discovery and functional analysis of monoterpenoid indole alkaloid pathways in plants. Methods Enzymol 2012, 515:207-229. 24. Nagatoshi M, Terasaka K, Nagatsu A, Mizukami H: Iridoidspecific glucosyltransferase from Gardenia jasminoides. J Biol Chem 2011, 286:32866-32874. Identifies the first known iridoid glucosyltransferase gene and opens the way to identify glucosyltransferases from other species of iridoid/MIA producing plants. 25. Murata J, Roepke J, Gordon H, De Luca V: The leaf epidermome of Catharanthus roseus reveals its biochemical specialization. Plant Cell 2008, 20:524-542. The study validates the use of carborundum abrasion for harvesting RNA from the epidermis of plant leaves and for producing representative transcriptomes of pathways enriched in this cell type. 26. De Luca V: Monoterpenoid indole alkaloid biosynthesis. In Plant Metabolism and Biotechnology, edn 1. Edited by Ashihara H, Crozier A, Komamine A.2011:263-291. 27. Besseau S, Kellner F, Lanoue A, Thamm AMK, Salim V, Schneider B, Geu-Flores B, Ho¨fer R, Guirimand G, Guihur A, Oudin A, Glevarec G, Foureau E, Papon N, Clastre M, GiglioliGuivarc’h N, St-Pierre B, Werck-Reichhart D, Burlat V, De Luca V, O’Connor SE, Courdavault V: A pair of tabersonine 16hydroxylases initiates the synthesis of vindoline in an organdependent manner in Catharanthus roseus. Plant Physiol 2013, 163:1792-1803. The study shows the importance of harvesting genes from organs actually producing a given metabolite for identifying genes actually involve in its biosynthesis. 28. Roepke J, Salim V, Thamm AJK, Murata J, Ploss K, Boland W, De Luca V: Vinca drug components accumulate exclusively in leaf exudates of Madagascar periwinkle. Proc Natl Acad Sci U S A 2010, 107:15287-15292. The study explains that low levels of anticancer drugs are made in Catharanthus roseus because the plant sequesters the precursors in separate locations. It also suggests the MIA biosynthesis in plants may have evolved with secretory processes that ensure their presence on the above ground plant surfaces where they accomplished unknown biological roles. 29. Yu F, De Luca V: ATP-binding cassette transporter controls leaf surface secretion of anticancer drug components in Current Opinion in Plant Biology 2014, 19:35–42
42 Physiology and metabolism
Catharanthus roseus. Proc Natl Acad Sci U S A 2013, 110:1583015835. The secretion of catharanthine and perhaps other MIAs was shown to be mediated by a plasma membrane associated transporter related to a common transporter involved in cuticle formation. This type of transporter was also identified in other MIA producing plant species and provides support to promote further studies on the secretory nature of MIA as a common feature of MIA accumulating plant species. 30. Carqueijeiro I, Noronha H, Duarte P, Gero´s H, Sottomayor M: Vacuolar transport of the medicinal alkaloids from Catharanthus roseus is mediated by a proton-driven antiport. Plant Physiol 2013, 162:1486-1496. A tonoplast associated proton-driven antiporter is carefully characterized at the biochemical level, providing some important insights for the mobilization and storage of vindoline in Catharanthus leaves. The study explores some controversial aspects of MIA transport in relation to the complexities involved.
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