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A survival strategy: The coevolution of the camptothecin biosynthetic pathway and self-resistance mechanism
Phytochemistry 70 (2009) 1894–1898
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Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Review
A survival strategy: The coevolution of the camptothecin biosynthetic pathway and self-resistance mechanism Supaart Sirikantaramas a, Mami Yamazaki b,c, Kazuki Saito b,d,* a
Plant Biochemistry Laboratory, Department of Plant Biology and Biotechnology, VKR Research Centre for Pro-Active Plants, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark b Graduate School of Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba 263-8522, Japan c Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawagichi, Saitama 332-0012, Japan d RIKEN Plant Science Center, Tsurumi-ku, Yokohama 230-0045, Japan
a r t i c l e
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Article history: Received 14 May 2009 Received in revised form 29 July 2009 Available online 24 August 2009 Keywords: Camptothecin-producing plants Camptothecin DNA topoisomerase 1 Self-resistance mechanism Coevolution
a b s t r a c t A diverse array of secondary metabolites in plants represents the process of coevolution between the plants and their natural enemies including herbivores and pathogens. For defense, plants produce many toxic compounds that harm other organisms. However, if the target of these compounds is a fundamental biological process then the producing plant may also be harmed. In such cases self-resistance strategies must coevolve with the biosynthetic pathway of toxic metabolites. In this review, we discuss the recent elucidation of the self-resistance mechanism of camptothecin (CPT)-producing plants. In this case the target protein of CPT, topoisomerase (Top) 1, has been mutated in order to overcome the toxicity of the compound. Similar mechanisms might also be used by other plants producing different toxic compounds which target fundamental metabolism. Ó 2009 Elsevier Ltd. All rights reserved.
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Coevolution between biosynthetic pathway and resistance mechanism Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Plants, as sessile organisms, have evolved complex mechanisms to protect themselves against insects and pathogens. The coevolution between plants and their natural enemies has been proposed to be a driving force for metabolic diversity, resulting in an estimated 200,000 secondary metabolites in plants (Hartmann, 2004). A combination of gene duplication, neofunctionalization, and positive selection are the mechanisms for the evolution of this diversity (Benderoth et al., 2006).
* Corresponding author. Address: Graduate School of Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba 263-8522, Japan. Tel.: +81 43 290 2904; fax: +81 43 290 2905. E-mail address: [email protected] (K. Saito). 0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2009.07.034
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In many cases, secondary metabolites and their pathway intermediates are phytotoxic. Plants have evolved several different mechanisms to avoid self-toxicity from different types of defense related secondary metabolites (Table 1) (see review, Sirikantaramas et al., 2008a). A basic strategy to avoid self-toxicity is tight regulation of biosynthesis in terms of temporal/spatial gene expression and localization. In this way secondary metabolites are biosynthesized under strict developmental control and accumulate in specific cell types or cellular compartments. Toxic cannabinoids are biosynthesized and accumulated in an extracellular storage cavity of glandular trichomes on leaves of Cannabis sativa (Sirikantaramas et al., 2005). This mechanism of cellular/ subcellular of harmful compounds is also applied to volatile compounds stored in glandular trichome in herbs such as peppermint (Dudareva et al., 2004). Diterpene sclareol is secreted to the extracellular space by a plasma membrane ATP-binding cassette (ABC)
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S. Sirikantaramas et al. / Phytochemistry 70 (2009) 1894–1898 Table 1 Reported self-resistance mechanism of toxic self-produced metabolites in plants. Compound
Self-resistance mechanism
Reference
Cannabinoids Volatiles
Extracellular biosynthesis Production in glandular trichomes Vacuolar sequestration Vacuolar sequestration Vacuolar sequestration and excretion Secretion by a ABC transporter Glycosylation Glycosylation
Sirikantaramas et al. (2005) Dudareva et al. (2004)
Glycosylation Glycosylation
Kliebenstein et al. (2005) Field and Osbourn (2008)
Mutation of target protein
Sirikantaramas et al. (2008b)
Flavonoids Berberine Sanguinarine Diterpene sclareol Benzoxazinoids Cyanogenic glycosides Glucosinolates Triterpenoid saponin Camptothecin
Rea (2007) Sakai et al. (2002) Alcantara et al. (2005) Jasinski et al. (2001) Osbourn (1996) Huges (1999)
transporter (Jasinski et al., 2001). Flavonoids are sequestered within vacuoles (for review, see Rea, 2007) and many alkaloids are also stored in the vacuole, so protecting the cell against their toxic effects (Martinoia et al., 2000; Wink and Roberts, 1998; Sakai et al., 2002; Alcantara et al., 2005). The involvement of transporters including ABC transporters and multidrug and toxic compound extrusion (MATE) transporters in flavonoid and alkaloid transport has also been reported (Goodman et al., 2004; Debeaujon et al., 2001; Marinova et al., 2007; Otani et al., 2005). Another common strategy is to modify toxic compounds into a stable and inactive form, for example benzoxazinoids, cyanogenic glycosides, glucosinolates, and triterpenoid saponins, are modified into a non-toxic form via glycosylation (Osbourn, 1996; Huges, 1999; Conn, 1980; Kliebenstein et al., 2005; Field and Osbourn, 2008). Recently, the discovery of gene clustering of plant secondary metabolite pathway has been discussed (Field and Osbourn, 2008; Bednarek and Osbourn, 2009). It has been known that genes for metabolic pathway are generally unclustered. However, the operon-like gene clusters for triterpene synthesis have been discovered in oat and Arabidopsis (Qi et al., 2004; Field and Osbourn, 2008). The disruption of the metabolic gene clusters can lead to accumulation of deleterious intermediates affecting the plant growth (Mylona et al., 2008; Field and Osbourn, 2008). Therefore, gene clustering might ensure plants that genes that are required for self-protection (i.e. glycosylation and/or sequestration) are co-inherited along with the genes for the pathway. There are many more examples of highly phytotoxic metabolites for which the self-resistance mechanisms are unknown. This is particularly so for medicinal plants which produce compounds that often interfere with basic biological processes. A very efficient self-resistance mechanism should coevolve with the biosynthesis of these compounds. Recently, we elucidated a novel self-resistance mechanism, in which a highly conserved target protein of a toxic compound is mutated to become resistant (Sirikantaramas et al., 2008b). In this case plants produce camptothecin (CPT), the terpenoid indole alkaloid that interferes with the function of eukaryotic DNA topoisomerase 1 (Top1). In the CPT-producing plants the Top1 gene is mutated and the activity of the encoded enzyme is unaffected by CPT. In this review, we further describe the target mutation-based mechanism for self-resistance to endogenously-produced toxic compounds and the adaptive coevolution between the CPT production system and its target Top1 in the producing plants. The same mechanism has also been found in CPT-resistant human cancer cells. We discuss whether similar mechanisms of target based mutation may also be found in other medicinal plants. If so the discovery of the target site mutations could be used to predict new emerging mutations in human cancer cells.
2. Coevolution between biosynthetic pathway and resistance mechanism in CPT-producing plants CPT is produced in several plants including Camptotheca acuminata, Nothapodytes foetida, and several species belonging to Ophiorrhiza genus. It is interesting to note that CPT-producing plants belong to different unrelated families – Apocynaceae, Icacinaceae, Nyssaceae, and Rubiaceae. Its semi-synthetic water-soluble CPT analogues, namely topotecan and irinotecan, are currently used as clinical anticancer drugs (for review, see Sirikantaramas et al., 2007a). While CPT accumulates in many different organs, the highest accumulation has been found in young leaves, flower buds, and roots (Yamazaki et al., 2003; López-Meyer et al., 1994). These parts are important for reproduction and growth. Because CPT is toxic to eukaryotic Top1 and the lack of observed insect damage to C. acuminata plantation in the USA has been reported (Liu et al., 1998), CPT is therefore beneficial to the plant for the prevention from insect attack. Because of the high toxicity of CPT, the production and detoxification must be well-coordinated in the producing plants. The hairy roots of Ophiorrhiza pumila have been used as a system to study the biosynthesis (Yamazaki et al., 2004) and the transport process of CPT (Sirikantaramas et al., 2007b). The hairy root accumulates a high level of CPT in the vacuole and also secretes it into the medium in a large quantity (Saito et al., 2001; Sudo et al., 2002). Both vacuolar sequestration and secretion were considered to be the mechanism to avoid the toxicity. However, we found that CPT secretion is a passive process depending on the concentration gradient between intracellular and extracellular compartments (Sirikantaramas et al., 2007b). This suggested there was another mechanism to enable the plants to survive in the presence of cytosolic CPT which could interfere with the function of Top1 in the nucleus. A lead was provided by the report that a mutation in Top1 confers CPT resistance in human cancer cells (Rasheed and Rubin, 2003). This led Sirikantaramas et al. (2008b) to investigate the structure of Top1 from CPT-producing plants. The study revealed a target mutation-based mechanism in Top1 which is the first example of self-resistance to endogenous toxic compounds employed by plants. Three point mutations in Top1 from CPT-producing plants have been identified. Surprisingly, one of the critical point mutations identified in Top1, Asn-722 to Ser722 (number according to human Top1), is identical to that found in CPT-resistant human leukemia cells (Fujimori et al., 1995). This position is the adjacent amino acid to the catalytically-functional Tyr-723, and this Asn-722 plays a role to hold CPT in the correct orientation in the catalytic pocket of Top1. This mutation is caused by the single nucleotide substitution of the second base of the codon, AAT (Asn) to AGT (Ser), both in CPT-producing plants and resistance human leukemia cells, implying the same selection pressure caused by CPT. The other two mutations found in plant Top1 have not yet been found in resistant human cancer cells but may possibly occur in the future. It is possible that these mutations are only found in plants because of the much longer evolutionary period of exposure to CPT in plants than in human cells.
Residue number 421
530
722
O. pumila
N
I
S
O. liukiuensis
N
I
S
C. acuminata
K
L
S
Fig. 1. Amino acid polymorphism in Top1s of CPT-producing plants. The underlined residues confer CPT resistance. The numbering is based on human Top1.
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Although all five CPT-producing plants investigated, namely C. acuminata, N. foetida, O. pumila, Ophiorrhiza liukiuensis, and Ophiorrhiza alata, belong to different families, they share the same resistance mechanism (i.e. target-based resistance) suggesting convergent evolution of this form of CPT resistance (Sirikantaramas et al., 2008b; unpublished results). Interestingly, Top1s of each genus posses different point mutations (Fig. 1). Since the activation of CPT biosynthetic pathway and the target-based resistance should be coevolved, this suggests that both processes must have developed after the speciation of these plants. Therefore, the CPT biosynthetic capacity in these distantly related plants could be due to convergent evolution. It is also possible that genes encoding biosynthetic enzymes have evolved early during evolution and they are not lost during phylogeny but might be active at some later point after the speciation (Wink and Witte, 1983; Wink, 2003, 2008). The Ophiorrhiza genus is composed of both CPT-producing and non-producing species. This provides a great benefit to follow the coevolution of the CPT biosynthetic pathway and Top1 mutation as a self-resistance mechanism. Interestingly, the non-producing Ophirrhiza japonica Top1 exhibits a partial resistance to CPT as determined by an in vivo CPT sensitivity assay in yeast, despite having no point mutations which are critical for resistance found in other CPT-producing plants. This suggests the involvement of other point mutation(s) which are responsible for Top1 pre-adaptation in O. japonica. Sirikantaramas et al. (2008) proposed a scheme for coevolution between the biosynthetic pathway and Top1 mutation-based resistance mechanism (Fig. 2). The scheme implies that Ophiorrhiza Top1 would have been pre-mutated, probably caused by an unknown intermediate (X) in the CPT biosynthetic pathway, resulting in plants that are primed for a certain amount of CPT. After the speciation of Ophiorrhiza, those with the CPT production ability would start producing CPT in a small amount that would not affect the plant survival. Higher production of CPT might have evolved later when the necessary mutations of Top1 have been triggered for the full resistance. To reinforce this scenario, it would be interesting to demonstrate the effect of the intermediates in the pathway on Top1 function; however, most of the intermediates in the pathway leading to CPT biosynthesis remain unknown. 3. Perspectives The finding of a target mutation-based mechanism in CPTproducing plants not only sheds light on the evolution of plant
secondary products but also the prediction of new emerging mutations in cancer cells. It might be a more efficient strategy and common mechanism than has previously been considered and is analagous to the strategy often found in organisms exhibiting resistance to exogenous compounds such as drugs and herbicides. It has been a long-standing question how plants are able to produce toxic metabolites without harming themselves. On the other hand, this question has been addressed recently in bacteria which produce toxic antibiotics (Hopwood, 2007). Tahlan et al. (2007) described the evolutionally primed resistance mechanism exhibited in bacterial antibiotic exporters. This could be a general mechanism in bacteria to prepare for resistance. To address this question, we need to understand how toxic metabolites are biosynthesized in terms of biosynthetic enzymes involved, localization, and transport. The advances in genome biology enable more opportunities to solve this question for many interesting compounds from a variety of medicinal plants and is particularly interesting for those compounds that have application as anticancer drugs. The properties of anticancer compounds are commonly able to interfere with the functions of basic biological units such as Top1, Top2, tubulin, and calcium pump. Less is known about which strategy producing plants use to cope with those compounds. What mechanism does Rauwolfia serpentina use to detoxify several alkaloids that exhibit inhibitory activities on either Top1 or Top2? (Itoh et al., 2005). A point mutation of tubulin coding gene has been reported to confer paclitaxel resistance in human cancer cells (Giannakakou et al., 1997). Is it possible that the same mutation mechanism also contributes to resistance in plants producing these antimitotic compounds including paclitaxel in Taxus brevifolia, podophyllotoxin in Podophyllum spp., vinblastine and vincristine in Catharanthus roseus, and colchicine in Colchicum autumnale? What mechanism does Thapsia garganica use to escape the toxicity of thapsigargin which shows inhibitory effect on golgi apparatuslocalized calcium pump in other plants such as a cauliflower and pea? (Ordenes et al., 2002). Glycosylation is unlikely to be the detoxification process because no glycosylated end products have been reported. Indole alkaloid vinblastine and vincristine are believed to be accumulated in the vacuole, however, there is no direct evidence to suggest this is the way to detoxify these compounds. Addressing these questions should give insight into the evolutional strategy of plants which produce toxic compounds for defense. This may also provide us a better understanding toward anticancer drug resistance and enable an improvement in design of potent drugs for human diseases such as cancer.
Fig. 2. Proposed coevolution between the CPT biosynthetic pathway and Top1 as CPT self-resistance mechanism. Strictosidine and strictosamide are the only intermediates in the pathway that have been identified. The black dots indicate mutation events caused by an unidentified intermediate (X) and CPT that confer partial and complete resistance to CPT, respectively.
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Acknowledgements The authors thanks for the financial supports by the Grants-inAid for Scientific Research from the Japan Society for the Promotion of Science and by CREST of the Japan Science and Technology Agency. We also thank Dr. Suchada Sukrong for sharing unpublished data, and Dr. Adam Takos for helpful discussion and proofreading of the manuscript. References Alcantara, J., Bird, D.A., Franceschi, V.R., Facchini, J., 2005. Sanguinarine biosynthesis is associated with the endoplasmic reticulum in cultured opium poppy cells after elicitor treatment. Plant Physiol. 138, 173–183. Bednarek, P., Osbourn, A., 2009. Plant-microbe interactions: chemical diversity in plant defense. Science 324, 746–748. Benderoth, M., Textor, S., Windsor, A.J., Mitchell-Olds, T., Gershenzon, J., Kroymann, J., 2006. Positive selection driving diversification in plant secondary metabolism. Proc. Natl. Acad. Sci. USA 103, 9118–9123. Conn, E.E., 1980. Cyanogenic glycosides. Annu. Rev. 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Supaart Sirikantaramas graduated from Chulalongkorn University, Thailand. He received the Japanese government scholarship for his MSc and PhD studies. In 2005, he obtained his PhD on the study of cannabinoid synthases from Cannabis sativa from Kyushu University, Japan, under the supervision of Prof. Yukihiro Shoyama. After that he worked as a postdoctoral fellow of the Japan Society for the Promotion of Science at Chiba University with Assoc. Prof. Mami Yamazaki and Prof. Kazuki Saito. There he investigated the transport and resistance mechanism of camptothecin in the producing plants. Since 2008, he has been working as a postdoc in the lab of Prof. Birger Lindberg Møller at the University of Copenhagen, Denmark, on Arabidopsis oxidative stress response.
Mami Yamazaki graduated from the Faculty of Pharmaceutical Sciences, Chiba University, Japan, in 1986 and then obtained her PhD for Pharmacognosy/Pharmaceutical Sciences from Chiba University in 1991. She received fellowship from Japan Society for the Promotion of Science (JSPS) for young scientists in 1990 for 2 years. She joined to Prof. Saito’s group as a faculty member since 1992 and has been promoted as Assoc. Prof. since 2000. During this period, she visited Ghent University under the sponsorship of JSPS for Research at Center of Excellence Abroad. She has spent busy days as a program officer in Ministry of Education, Culture, Sports, Science, and Technology from 2006 to 2008. She is interested in evolution and diversity of specialized metabolisms in plants.
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Kazuki Saito graduated from the Faculty of Pharmaceutical Sciences, the University of Tokyo, Japan, in 1977 and then obtained his PhD for bio-organic chemistry/ biochemistry of pharmaceutical sciences from the University of Tokyo in 1982. After staying in Keio University in Japan and Ghent University in Belgium (Prof. Marc Van Montagu’s laboratory), he became a faculty member at the Graduate School of Pharmaceutical Sciences, Chiba University, Japan. There he has been appointed as a full professor since 1995, until now. Since April, 2005, he has been additionally appointed as a group director at RIKEN Plant Science Center in Yokohama to direct Metabolomics Research group and Metabolic Function Group. He has published more than 200 original papers and 80 invited reviews and book chapters. His
research interests are metabolome-based functional genomics, biochemistry, molecular biology and biotechnology of primary and secondary metabolism in plants. In particular, he is engaging in the biosynthetic studies of sulfur compounds, flavonoids, terpenoids and alkaloids by means of metabolomics.
Contents lists available at ScienceDirect
Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Review
A survival strategy: The coevolution of the camptothecin biosynthetic pathway and self-resistance mechanism Supaart Sirikantaramas a, Mami Yamazaki b,c, Kazuki Saito b,d,* a
Plant Biochemistry Laboratory, Department of Plant Biology and Biotechnology, VKR Research Centre for Pro-Active Plants, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark b Graduate School of Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba 263-8522, Japan c Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawagichi, Saitama 332-0012, Japan d RIKEN Plant Science Center, Tsurumi-ku, Yokohama 230-0045, Japan
a r t i c l e
i n f o
Article history: Received 14 May 2009 Received in revised form 29 July 2009 Available online 24 August 2009 Keywords: Camptothecin-producing plants Camptothecin DNA topoisomerase 1 Self-resistance mechanism Coevolution
a b s t r a c t A diverse array of secondary metabolites in plants represents the process of coevolution between the plants and their natural enemies including herbivores and pathogens. For defense, plants produce many toxic compounds that harm other organisms. However, if the target of these compounds is a fundamental biological process then the producing plant may also be harmed. In such cases self-resistance strategies must coevolve with the biosynthetic pathway of toxic metabolites. In this review, we discuss the recent elucidation of the self-resistance mechanism of camptothecin (CPT)-producing plants. In this case the target protein of CPT, topoisomerase (Top) 1, has been mutated in order to overcome the toxicity of the compound. Similar mechanisms might also be used by other plants producing different toxic compounds which target fundamental metabolism. Ó 2009 Elsevier Ltd. All rights reserved.
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Coevolution between biosynthetic pathway and resistance mechanism Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Plants, as sessile organisms, have evolved complex mechanisms to protect themselves against insects and pathogens. The coevolution between plants and their natural enemies has been proposed to be a driving force for metabolic diversity, resulting in an estimated 200,000 secondary metabolites in plants (Hartmann, 2004). A combination of gene duplication, neofunctionalization, and positive selection are the mechanisms for the evolution of this diversity (Benderoth et al., 2006).
* Corresponding author. Address: Graduate School of Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba 263-8522, Japan. Tel.: +81 43 290 2904; fax: +81 43 290 2905. E-mail address: [email protected] (K. Saito). 0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2009.07.034
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In many cases, secondary metabolites and their pathway intermediates are phytotoxic. Plants have evolved several different mechanisms to avoid self-toxicity from different types of defense related secondary metabolites (Table 1) (see review, Sirikantaramas et al., 2008a). A basic strategy to avoid self-toxicity is tight regulation of biosynthesis in terms of temporal/spatial gene expression and localization. In this way secondary metabolites are biosynthesized under strict developmental control and accumulate in specific cell types or cellular compartments. Toxic cannabinoids are biosynthesized and accumulated in an extracellular storage cavity of glandular trichomes on leaves of Cannabis sativa (Sirikantaramas et al., 2005). This mechanism of cellular/ subcellular of harmful compounds is also applied to volatile compounds stored in glandular trichome in herbs such as peppermint (Dudareva et al., 2004). Diterpene sclareol is secreted to the extracellular space by a plasma membrane ATP-binding cassette (ABC)
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S. Sirikantaramas et al. / Phytochemistry 70 (2009) 1894–1898 Table 1 Reported self-resistance mechanism of toxic self-produced metabolites in plants. Compound
Self-resistance mechanism
Reference
Cannabinoids Volatiles
Extracellular biosynthesis Production in glandular trichomes Vacuolar sequestration Vacuolar sequestration Vacuolar sequestration and excretion Secretion by a ABC transporter Glycosylation Glycosylation
Sirikantaramas et al. (2005) Dudareva et al. (2004)
Glycosylation Glycosylation
Kliebenstein et al. (2005) Field and Osbourn (2008)
Mutation of target protein
Sirikantaramas et al. (2008b)
Flavonoids Berberine Sanguinarine Diterpene sclareol Benzoxazinoids Cyanogenic glycosides Glucosinolates Triterpenoid saponin Camptothecin
Rea (2007) Sakai et al. (2002) Alcantara et al. (2005) Jasinski et al. (2001) Osbourn (1996) Huges (1999)
transporter (Jasinski et al., 2001). Flavonoids are sequestered within vacuoles (for review, see Rea, 2007) and many alkaloids are also stored in the vacuole, so protecting the cell against their toxic effects (Martinoia et al., 2000; Wink and Roberts, 1998; Sakai et al., 2002; Alcantara et al., 2005). The involvement of transporters including ABC transporters and multidrug and toxic compound extrusion (MATE) transporters in flavonoid and alkaloid transport has also been reported (Goodman et al., 2004; Debeaujon et al., 2001; Marinova et al., 2007; Otani et al., 2005). Another common strategy is to modify toxic compounds into a stable and inactive form, for example benzoxazinoids, cyanogenic glycosides, glucosinolates, and triterpenoid saponins, are modified into a non-toxic form via glycosylation (Osbourn, 1996; Huges, 1999; Conn, 1980; Kliebenstein et al., 2005; Field and Osbourn, 2008). Recently, the discovery of gene clustering of plant secondary metabolite pathway has been discussed (Field and Osbourn, 2008; Bednarek and Osbourn, 2009). It has been known that genes for metabolic pathway are generally unclustered. However, the operon-like gene clusters for triterpene synthesis have been discovered in oat and Arabidopsis (Qi et al., 2004; Field and Osbourn, 2008). The disruption of the metabolic gene clusters can lead to accumulation of deleterious intermediates affecting the plant growth (Mylona et al., 2008; Field and Osbourn, 2008). Therefore, gene clustering might ensure plants that genes that are required for self-protection (i.e. glycosylation and/or sequestration) are co-inherited along with the genes for the pathway. There are many more examples of highly phytotoxic metabolites for which the self-resistance mechanisms are unknown. This is particularly so for medicinal plants which produce compounds that often interfere with basic biological processes. A very efficient self-resistance mechanism should coevolve with the biosynthesis of these compounds. Recently, we elucidated a novel self-resistance mechanism, in which a highly conserved target protein of a toxic compound is mutated to become resistant (Sirikantaramas et al., 2008b). In this case plants produce camptothecin (CPT), the terpenoid indole alkaloid that interferes with the function of eukaryotic DNA topoisomerase 1 (Top1). In the CPT-producing plants the Top1 gene is mutated and the activity of the encoded enzyme is unaffected by CPT. In this review, we further describe the target mutation-based mechanism for self-resistance to endogenously-produced toxic compounds and the adaptive coevolution between the CPT production system and its target Top1 in the producing plants. The same mechanism has also been found in CPT-resistant human cancer cells. We discuss whether similar mechanisms of target based mutation may also be found in other medicinal plants. If so the discovery of the target site mutations could be used to predict new emerging mutations in human cancer cells.
2. Coevolution between biosynthetic pathway and resistance mechanism in CPT-producing plants CPT is produced in several plants including Camptotheca acuminata, Nothapodytes foetida, and several species belonging to Ophiorrhiza genus. It is interesting to note that CPT-producing plants belong to different unrelated families – Apocynaceae, Icacinaceae, Nyssaceae, and Rubiaceae. Its semi-synthetic water-soluble CPT analogues, namely topotecan and irinotecan, are currently used as clinical anticancer drugs (for review, see Sirikantaramas et al., 2007a). While CPT accumulates in many different organs, the highest accumulation has been found in young leaves, flower buds, and roots (Yamazaki et al., 2003; López-Meyer et al., 1994). These parts are important for reproduction and growth. Because CPT is toxic to eukaryotic Top1 and the lack of observed insect damage to C. acuminata plantation in the USA has been reported (Liu et al., 1998), CPT is therefore beneficial to the plant for the prevention from insect attack. Because of the high toxicity of CPT, the production and detoxification must be well-coordinated in the producing plants. The hairy roots of Ophiorrhiza pumila have been used as a system to study the biosynthesis (Yamazaki et al., 2004) and the transport process of CPT (Sirikantaramas et al., 2007b). The hairy root accumulates a high level of CPT in the vacuole and also secretes it into the medium in a large quantity (Saito et al., 2001; Sudo et al., 2002). Both vacuolar sequestration and secretion were considered to be the mechanism to avoid the toxicity. However, we found that CPT secretion is a passive process depending on the concentration gradient between intracellular and extracellular compartments (Sirikantaramas et al., 2007b). This suggested there was another mechanism to enable the plants to survive in the presence of cytosolic CPT which could interfere with the function of Top1 in the nucleus. A lead was provided by the report that a mutation in Top1 confers CPT resistance in human cancer cells (Rasheed and Rubin, 2003). This led Sirikantaramas et al. (2008b) to investigate the structure of Top1 from CPT-producing plants. The study revealed a target mutation-based mechanism in Top1 which is the first example of self-resistance to endogenous toxic compounds employed by plants. Three point mutations in Top1 from CPT-producing plants have been identified. Surprisingly, one of the critical point mutations identified in Top1, Asn-722 to Ser722 (number according to human Top1), is identical to that found in CPT-resistant human leukemia cells (Fujimori et al., 1995). This position is the adjacent amino acid to the catalytically-functional Tyr-723, and this Asn-722 plays a role to hold CPT in the correct orientation in the catalytic pocket of Top1. This mutation is caused by the single nucleotide substitution of the second base of the codon, AAT (Asn) to AGT (Ser), both in CPT-producing plants and resistance human leukemia cells, implying the same selection pressure caused by CPT. The other two mutations found in plant Top1 have not yet been found in resistant human cancer cells but may possibly occur in the future. It is possible that these mutations are only found in plants because of the much longer evolutionary period of exposure to CPT in plants than in human cells.
Residue number 421
530
722
O. pumila
N
I
S
O. liukiuensis
N
I
S
C. acuminata
K
L
S
Fig. 1. Amino acid polymorphism in Top1s of CPT-producing plants. The underlined residues confer CPT resistance. The numbering is based on human Top1.
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Although all five CPT-producing plants investigated, namely C. acuminata, N. foetida, O. pumila, Ophiorrhiza liukiuensis, and Ophiorrhiza alata, belong to different families, they share the same resistance mechanism (i.e. target-based resistance) suggesting convergent evolution of this form of CPT resistance (Sirikantaramas et al., 2008b; unpublished results). Interestingly, Top1s of each genus posses different point mutations (Fig. 1). Since the activation of CPT biosynthetic pathway and the target-based resistance should be coevolved, this suggests that both processes must have developed after the speciation of these plants. Therefore, the CPT biosynthetic capacity in these distantly related plants could be due to convergent evolution. It is also possible that genes encoding biosynthetic enzymes have evolved early during evolution and they are not lost during phylogeny but might be active at some later point after the speciation (Wink and Witte, 1983; Wink, 2003, 2008). The Ophiorrhiza genus is composed of both CPT-producing and non-producing species. This provides a great benefit to follow the coevolution of the CPT biosynthetic pathway and Top1 mutation as a self-resistance mechanism. Interestingly, the non-producing Ophirrhiza japonica Top1 exhibits a partial resistance to CPT as determined by an in vivo CPT sensitivity assay in yeast, despite having no point mutations which are critical for resistance found in other CPT-producing plants. This suggests the involvement of other point mutation(s) which are responsible for Top1 pre-adaptation in O. japonica. Sirikantaramas et al. (2008) proposed a scheme for coevolution between the biosynthetic pathway and Top1 mutation-based resistance mechanism (Fig. 2). The scheme implies that Ophiorrhiza Top1 would have been pre-mutated, probably caused by an unknown intermediate (X) in the CPT biosynthetic pathway, resulting in plants that are primed for a certain amount of CPT. After the speciation of Ophiorrhiza, those with the CPT production ability would start producing CPT in a small amount that would not affect the plant survival. Higher production of CPT might have evolved later when the necessary mutations of Top1 have been triggered for the full resistance. To reinforce this scenario, it would be interesting to demonstrate the effect of the intermediates in the pathway on Top1 function; however, most of the intermediates in the pathway leading to CPT biosynthesis remain unknown. 3. Perspectives The finding of a target mutation-based mechanism in CPTproducing plants not only sheds light on the evolution of plant
secondary products but also the prediction of new emerging mutations in cancer cells. It might be a more efficient strategy and common mechanism than has previously been considered and is analagous to the strategy often found in organisms exhibiting resistance to exogenous compounds such as drugs and herbicides. It has been a long-standing question how plants are able to produce toxic metabolites without harming themselves. On the other hand, this question has been addressed recently in bacteria which produce toxic antibiotics (Hopwood, 2007). Tahlan et al. (2007) described the evolutionally primed resistance mechanism exhibited in bacterial antibiotic exporters. This could be a general mechanism in bacteria to prepare for resistance. To address this question, we need to understand how toxic metabolites are biosynthesized in terms of biosynthetic enzymes involved, localization, and transport. The advances in genome biology enable more opportunities to solve this question for many interesting compounds from a variety of medicinal plants and is particularly interesting for those compounds that have application as anticancer drugs. The properties of anticancer compounds are commonly able to interfere with the functions of basic biological units such as Top1, Top2, tubulin, and calcium pump. Less is known about which strategy producing plants use to cope with those compounds. What mechanism does Rauwolfia serpentina use to detoxify several alkaloids that exhibit inhibitory activities on either Top1 or Top2? (Itoh et al., 2005). A point mutation of tubulin coding gene has been reported to confer paclitaxel resistance in human cancer cells (Giannakakou et al., 1997). Is it possible that the same mutation mechanism also contributes to resistance in plants producing these antimitotic compounds including paclitaxel in Taxus brevifolia, podophyllotoxin in Podophyllum spp., vinblastine and vincristine in Catharanthus roseus, and colchicine in Colchicum autumnale? What mechanism does Thapsia garganica use to escape the toxicity of thapsigargin which shows inhibitory effect on golgi apparatuslocalized calcium pump in other plants such as a cauliflower and pea? (Ordenes et al., 2002). Glycosylation is unlikely to be the detoxification process because no glycosylated end products have been reported. Indole alkaloid vinblastine and vincristine are believed to be accumulated in the vacuole, however, there is no direct evidence to suggest this is the way to detoxify these compounds. Addressing these questions should give insight into the evolutional strategy of plants which produce toxic compounds for defense. This may also provide us a better understanding toward anticancer drug resistance and enable an improvement in design of potent drugs for human diseases such as cancer.
Fig. 2. Proposed coevolution between the CPT biosynthetic pathway and Top1 as CPT self-resistance mechanism. Strictosidine and strictosamide are the only intermediates in the pathway that have been identified. The black dots indicate mutation events caused by an unidentified intermediate (X) and CPT that confer partial and complete resistance to CPT, respectively.
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Supaart Sirikantaramas graduated from Chulalongkorn University, Thailand. He received the Japanese government scholarship for his MSc and PhD studies. In 2005, he obtained his PhD on the study of cannabinoid synthases from Cannabis sativa from Kyushu University, Japan, under the supervision of Prof. Yukihiro Shoyama. After that he worked as a postdoctoral fellow of the Japan Society for the Promotion of Science at Chiba University with Assoc. Prof. Mami Yamazaki and Prof. Kazuki Saito. There he investigated the transport and resistance mechanism of camptothecin in the producing plants. Since 2008, he has been working as a postdoc in the lab of Prof. Birger Lindberg Møller at the University of Copenhagen, Denmark, on Arabidopsis oxidative stress response.
Mami Yamazaki graduated from the Faculty of Pharmaceutical Sciences, Chiba University, Japan, in 1986 and then obtained her PhD for Pharmacognosy/Pharmaceutical Sciences from Chiba University in 1991. She received fellowship from Japan Society for the Promotion of Science (JSPS) for young scientists in 1990 for 2 years. She joined to Prof. Saito’s group as a faculty member since 1992 and has been promoted as Assoc. Prof. since 2000. During this period, she visited Ghent University under the sponsorship of JSPS for Research at Center of Excellence Abroad. She has spent busy days as a program officer in Ministry of Education, Culture, Sports, Science, and Technology from 2006 to 2008. She is interested in evolution and diversity of specialized metabolisms in plants.
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Kazuki Saito graduated from the Faculty of Pharmaceutical Sciences, the University of Tokyo, Japan, in 1977 and then obtained his PhD for bio-organic chemistry/ biochemistry of pharmaceutical sciences from the University of Tokyo in 1982. After staying in Keio University in Japan and Ghent University in Belgium (Prof. Marc Van Montagu’s laboratory), he became a faculty member at the Graduate School of Pharmaceutical Sciences, Chiba University, Japan. There he has been appointed as a full professor since 1995, until now. Since April, 2005, he has been additionally appointed as a group director at RIKEN Plant Science Center in Yokohama to direct Metabolomics Research group and Metabolic Function Group. He has published more than 200 original papers and 80 invited reviews and book chapters. His
research interests are metabolome-based functional genomics, biochemistry, molecular biology and biotechnology of primary and secondary metabolism in plants. In particular, he is engaging in the biosynthetic studies of sulfur compounds, flavonoids, terpenoids and alkaloids by means of metabolomics.