Erysimum Cheiranthoides, an Ecological Research System with Potential As a Genetic and Genomic Model for Studying Cardiac Glycoside Biosynthesis
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Phytochem Rev (2018) 17:1239–1251 https://doi.org/10.1007/s11101-018-9562-4 (0123456789().,-volV)(0123456789().,-volV) Erysimum cheiranthoides, an ecological research system with potential as a genetic and genomic model for studying cardiac glycoside biosynthesis Tobias Züst . Mahdieh Mirzaei . Georg Jander Received: 18 January 2018 / Accepted: 15 March 2018 / Published online: 20 March 2018 © Springer Science+Business Media B.V., part of Springer Nature 2018 Abstract At least twelve plant families contain defensive metabolites. We propose that Erysimum species that synthesize cardiac glycosides as defense cheiranthoides (wormseed wallflower), a rapid-cy- against herbivory. These inhibitors of animal Na+, cling, self-pollinating species with a relatively small, K+-ATPases also have medical uses in treating diploid genome, would be a suitable model system to congestive heart failure and other diseases. However, advance research on the biosynthesis of cardiac despite extensive ecological research and centuries of glycosides in plants. use in both traditional and modern medicine, the complete cardiac glycoside biosynthesis pathway has Keywords Erysimum cheiranthoides · yet to be elucidated in any plant species. To a large Wallflower · Cardiac glycoside · Cardenolide · extent, this research deficit results from the fact that Model system cardiac glycosides are produced exclusively by non- model plant species such as Digitalis that have not been amenable to the development of mutagenesis, Introduction cloning, and genetic mapping approaches. Recent advances in genome sequencing, transcript profiling, Cardiac glycosides are a diverse group of natural plant transformation, transient expression assays, and products that act as allosteric inhibitors of Na+,K+- plant metabolite analysis have provided new oppor- ATPase, an essential membrane ion transporter that is tunities for the investigation and elucidation of found in almost all animal cells. Broadly, cardiac cardiac glycoside biosynthesis pathways. The genetic glycosides can be categorized as cardenolides and tools that have been developed for Brassicaceae, in bufadienolides, which have a common steroid core particular Arabidopsis thaliana, may be directly (5β,14β-androstane-3β14-diol) and differ according applicable to Erysimum, a Brassicaceae genus that to the presence of a five- or six-membered lactone characteristically produces cardiac glycosides as ring. This steroid-lactone core structure is highly conserved among cardiac glycosides and mediates the specific binding of cardiac glycosides to Na+,K+- T. Zu¨st Institute of Plant Sciences, University of Bern, ATPase (Dzimiri et al. 1987). While some cardeno- Altenbergrain 21, 3013 Bern, Switzerland lides and bufadienolides naturally occur as aglycones (genins), most are linked to one or several sugar & M. Mirzaei · G. Jander ( ) moieties in a linear chain, resulting in a glycoside Boyce Thompson Institute, 533 Tower Road, Ithaca, NY 14853, USA ‘tail’ that significantly increases the binding affinity e-mail: [email protected] and inhibitory effect of these compounds (Dzimiri 123 1240 Phytochem Rev (2018) 17:1239–1251 et al. 1987). Several hundred cardenolide and bufa- this genus has significantly hindered progress in dienolide structures, which differ by substitutions to unravelling the full cardiac glycoside metabolic functional groups on the steroid core, stereoisomeric pathway. In contrast, the cardenolide-producing conformation, or the incorporation of different types Brassicaceae genus Erysimum is closely related to of sugars in the glycoside tail, have been described A. thaliana (Huang et al. 2016), and has been (Kreis and Mu¨ller-Uri 2010; Melero et al. 2000; proposed as a more suitable model for investigating Singh and Rastogi 1970). Although most cardiac the molecular biology of cardiac glycoside biosyn- glycosides have been identified and isolated from thesis (Munkert et al. 2011). Within this genus, plants, they are also found in certain insects and toads Erysimum cheiranthoides (wormseed wallflower; (hence the name bufadienolide). Cardiac glycosides Fig. 1), a rapid-cycling diploid species with a have no known function in the core metabolism of the relatively small genome size, would provide an plants that produce them, and thus likely act primarily excellent model system for the use of genetic and in defense against insects and other herbivores. genomic approaches to investigate the biosynthesis, The ability to produce cardiac glycosides is ecological function, and evolutionary origins of scattered across the plant phylogenetic tree, with at cardiac glycoside biosynthesis. Here, we provide an least a dozen plant families (Apocynaceae, Aspara- overview of currently known steps in cardiac gly- gaceae, Brassicaceae, Celastraceae, Crassulaceae, coside synthesis in general, and in Erysimum in Euphorbiaceae, Fabaceae, Malvaceae, Moraceae, particular. Additionally, we review chemical, eco- Plantaginaceae, Ranunculaceae, and Zingiberaceae) logical, evolutionary, and ethnobotanical literature on containing cardiac glycoside-producing species Erysimum to highlight its relevance as a model (Agrawal et al. 2012; Melero et al. 2000; Steyn and system in diverse research areas, and conclude by van Heerden 1998). In the Apocynaceae, cardiac outlining a set of research methods that would be glycosides are the predominant secondary metabo- required for developing E. cheiranthoides as a new lites in most species and likely represent an ancestral plant genetic and genomic model system. trait of this family. In the other plant families, there are only sporadic occurrences of cardiac glycoside in subclades or single known species, which likely Biosynthesis of cardiac glycosides represent cases of relatively recent, repeated pathway evolution. Such convergent evolution of plant toxins Production of cardenolides and bufadienolides likely with highly similar inhibitory effects on animal Na+, evolved from pathways for the biosynthesis of K+-ATPases suggests both a strong selective pressure phytosterols (24 alkyl sterols) and endogenous plant for plant defense against herbivory and a common steroid hormones, e.g. brassinosteroids. Early isotope metabolic origin that facilitates repeated evolution of labeling studies suggested that cardiac glycosides are similar or identical molecular structures. synthesized from cholesterol with progesterone as an The discovery of the metabolic pathways involved intermediate (Kreis et al. 1998; Theurer et al. 1994). in cardiac glycoside synthesis has been limited by the However, despite decades of research with Digitalis, fact that these compounds are not found in traditional only a relatively small number of enzymes in the genetic model species. The elucidation of the biosyn- cardenolide biosynthesis pathway have been identi- thetic pathways of other important plant metabolites fied. Progesterone 5β-reductase, which catalyzes the such as glucosinolates, benzoxazinoids, and flavo- conversion of progesterone to 5β-pregnane-3,20- noids was greatly facilitated by the genomic and dione (Fig. 2), was first cloned from Digitalis molecular resources available for model plants such purpurea (Ga¨rtner et al. 1994) and is expressed in as Arabidopsis thaliana, Zea mays, Oryza sativa, all tested Digitalis species (Kreis 2017). Two other Solanum lycopersicum, and Medicago truncatula. early steps in cardenolide biosynthesis, dehydrogena- Even though the biosynthesis of cardiac glycosides tion of pregnenolone to isoprogesterone and has been studied extensively in Digitalis spp. (fox- reduction of 5β-pregnane-3,20-dione (Fig. 2), are glove; Kreis 2017; Luckner and Wichtl 2000), the both catalyzed by 3β-hydroxysteroid dehydrogenases. relatively large plant size, complex pollination After their initial identification in Digitalis lanata requirement, and long, often biennial life cycle of (Finsterbusch et al. 1999; Herl et al. 2006), 3β- 123 Phytochem Rev (2018) 17:1239–1251 1241 A B C D Fig. 1 Erysimum cheiranthoides. a Natural growth in the Switzerland. c, d Plants after 2 and 6 weeks growth, Lenzen-Elbtalaue (Elbe River floodplain) in Germany. respectively, in a growth chamber with fluorescent lights b Growth in a disturbed habitat near the Aare River in Bern, hydroxysteroid dehydrogenases have been identified has been characterized enzymatically in D. purpurea and cloned from several other Digitalis species. (Kuate et al. 2008). Other enzymes of cardiac Given the relatively broad substrate specificities of glycoside biosynthesis, including those catalyzing the identified progesterone 5β-reductase and 3β- the addition of sugar side chains and those modifying hydroxysteroid dehydrogenase enzymes, it is quite the aglycone functional groups, remain to be possible that their in vivo substrates are not those that identified. are postulated in Fig. 2, but rather other endogenous The investigation of cardenolide biosynthetic plant steroids. Malonyl coenzyme A:21-hydroxypreg- enzymes in Erysimum is a direct extension of the nane 21-O-malonyltransferase, which is required for prior work that has been done with Digitalis. the synthesis of the cardenolide lactone ring (Fig. 2), Predicted progesterone 5β-reductase genes have been 123 1242 Phytochem Rev (2018) 17:1239–1251 plant steroid metabolism pregnenolone 5β-pregnane- 3β,14β-diol-20-one 14β-hydroxypregnane 3β-hydroxysteroid 21β-hydroxylase dehydrogenase isoprogesterone 5β-pregnane-3β,14β- 21-triiol-20-one malonyl coenzyme A:21- Δ5-Δ4-ketosteroid hydroxylpregnane 21-O- isomerase malonyltransferase