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Wolfgang Kreis - One of the best experts on this subject based on the ideXlab platform.
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rnai mediated gene knockdown of progesterone 5β reductases in digitalis lanata reduces 5β Cardenolide content
Plant Cell Reports, 2021Co-Authors: Jan Klein, Wolfgang Kreis, Mona Ernst, Elisa Horn, Tim Leykauf, Tamara Leupold, Maja Dorfner, Laura Wolf, Anastasiia Ignatova, Jennifer MunkertAbstract:Studying RNAi-mediated DlP5βR1 and DlP5βR2 knockdown shoot culture lines of Digitalis lanata, we here provide direct evidence for the participation of PRISEs (progesterone 5β-reductase/iridoid synthase-like enzymes) in 5β-Cardenolide formation. Progesterone 5β-reductases (P5βR) are assumed to catalyze the reduction of progesterone to 5β-pregnane-3,20-dione, which is a crucial step in the biosynthesis of the 5β-Cardenolides. P5βRs are encoded by VEP1-like genes occurring ubiquitously in embryophytes. P5βRs are substrate-promiscuous enone-1,4-reductases recently termed PRISEs (progesterone 5β-reductase/iridoid synthase-like enzymes). Two PRISE genes, termed DlP5βR1 (AY585867.1) and DlP5βR2 (HM210089.1) were isolated from Digitalis lanata. To give experimental evidence for the participation of PRISEs in 5β-Cardenolide formation, we here established several RNAi-mediated DlP5βR1 and DlP5βR2 knockdown shoot culture lines of D. lanata. Cardenolide contents were lower in D. lanata P5βR-RNAi lines than in wild-type shoots. We considered that the gene knockdowns may have had pleiotropic effects such as an increase in glutathione (GSH) which is known to inhibit Cardenolide formation. GSH levels and expression of glutathione reductase (GR) were measured. Both were higher in the Dl P5βR-RNAi lines than in the wild-type shoots. Cardenolide biosynthesis was restored by buthionine sulfoximine (BSO) treatment in Dl P5βR2-RNAi lines but not in Dl P5βR1-RNAi lines. Since progesterone is a precursor of Cardenolides but can also act as a reactive electrophile species (RES), we here discriminated between these by comparing the effects of progesterone and methyl vinyl ketone, a small RES but not a precursor of Cardenolides. To the best of our knowledge, we here demonstrated for the first time that P5βR1 is involved in Cardenolide formation. We also provide further evidence that PRISEs are also important for plants dealing with stress by detoxifying reactive electrophile species (RES).
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the new semisynthetic Cardenolide analog 3β 2 1 amantadine 1 on ethylamine digitoxigenin amantadig efficiently suppresses cell growth in human leukemia and urological tumor cell lines
Anticancer Research, 2015Co-Authors: Elke Nolte, Anna Sobel, Sven Wach, Heidi Hertlein, Nadja Ebert, Frieder Mulleruri, Robert K Slany, Helge Taubert, Bernd Wullich, Wolfgang KreisAbstract:The use of Cardenolides in the treatment of cardiac insufficiency is well-established. However, the potential of Cardenolides in tumor therapy has not been comprehensively studied. The aim of the present study was to characterize the cytotoxic effects of the new semisynthetic Cardenolide analog AMANTADIG (3β-[2-(1-amantadine)-1-on-ethylamine]-digitoxigenin), and the Cardenolide digitoxin on leukemia and urological tumor cell lines.The anti-proliferative effects of AMANTADIG and digitoxin on leukemia and urological cancer cell lines were analyzed using (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction viability assay.AMANTADIG and digitoxin exhibited anti-proliferative activities against the leukemia cell lines in the low nanomolar range. The prostate cancer and renal cell carcinoma cell lines were equally sensitive to AMANTADIG and digitoxin, however, the leukemia cell lines were more sensitive to both Cardenolides.The new Cardenolide analog AMANTADIG appears effective in cell growth inhibition of leukemia and urological tumor cell lines.
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the new semisynthetic Cardenolide analog 3β 2 1 amantadine 1 on ethylamine digitoxigenin amantadig efficiently suppresses cell growth in human leukemia and urological tumor cell lines
Anticancer Research, 2015Co-Authors: Elke Nolte, Anna Sobel, Sven Wach, Heidi Hertlein, Nadja Ebert, Frieder Mulleruri, Robert K Slany, Helge Taubert, Bernd Wullich, Wolfgang KreisAbstract:Background/aim The use of Cardenolides in the treatment of cardiac insufficiency is well-established. However, the potential of Cardenolides in tumor therapy has not been comprehensively studied. The aim of the present study was to characterize the cytotoxic effects of the new semisynthetic Cardenolide analog AMANTADIG (3β-[2-(1-amantadine)-1-on-ethylamine]-digitoxigenin), and the Cardenolide digitoxin on leukemia and urological tumor cell lines. Materials and methods The anti-proliferative effects of AMANTADIG and digitoxin on leukemia and urological cancer cell lines were analyzed using (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction viability assay. Results AMANTADIG and digitoxin exhibited anti-proliferative activities against the leukemia cell lines in the low nanomolar range. The prostate cancer and renal cell carcinoma cell lines were equally sensitive to AMANTADIG and digitoxin, however, the leukemia cell lines were more sensitive to both Cardenolides. Conclusion The new Cardenolide analog AMANTADIG appears effective in cell growth inhibition of leukemia and urological tumor cell lines.
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Cardenolide estimation in callus mediated regenerants of digitalis lamarckii ivanina dwarf foxglove
In Vitro Cellular & Developmental Biology – Plant, 2014Co-Authors: Buhara Yucesan, Wolfgang Kreis, Frieder Mulleruri, Ekrem GurelAbstract:Digitalis Cardenolides can regulate heart rhythms and are effective agents in cancer chemotherapy, in particular, for treating prostate and breast cancer. In this study, an optimized and efficient plant tissue culture protocol was established using callus cultures of Digitalis lamarckii Ivanina, commonly known as dwarf foxglove. Lamina explants developed callus when cultured on Linsmaier and Skoog (LS) medium containing different concentrations of 6-benzyladenine (BA; 4.4, 13.3, or 22.2 μM) and α-naphthalene acetic acid (NAA; 2.7, 5.4, or 10.8 μM). The highest incidence of callus formation (100%) was achieved on LS medium containing 13.3 μM BA and 10.8 μM NAA. Indirect shoot regeneration was achieved when the callus explants were cultured on LS medium supplemented with varying concentrations of BA (0.4, 1.1, or 2.2 μM) and/or gibberellic acid (0.7 or 1.4 μM) for 8 wk. Following the rooting of shoots on LS medium supplemented with either indole-3-acetic acid (ranging from 1.4 to 5.7 μM) or NAA (1.3 to 5.2 μM), lamina and petiole tissues of the 4-mo-old regenerated plants were compared for their Cardenolide contents. Lamina extracts showed nearly three times higher Cardenolide accumulation than petiole extracts. Of the Cardenolides analyzed by reverse-phase high-performance liquid chromatography, neo-odorobioside G and glucogitoroside were abundant in lamina extracts (170.3 and 143.9 mg/kg dry weight, respectively). The regeneration protocol described in this study can be used for the in vitro production of certain Cardenolides from D. lamarckii.
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expression of 3β hsd and p5βr genes respectively coding for δ5 3β hydroxysteroid dehydrogenase and progesterone 5β reductase in leaves and cell cultures of digitalis lanata ehrh
Planta Medica, 2010Co-Authors: Mona Ernst, Frieder Mulleruri, Rodrigo Maia De Padua, Vanessa Herl, Wolfgang KreisAbstract:Plants of the genus Digitalis produce 5 beta-Cardenolides that are used in the therapy of cardiac insufficiency in humans. 3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) and progesterone 5 beta-reductase (P5 betaR) are both supposed to be important enzymes in the biosynthesis of these natural products. Activity and gene expression were demonstrated for both enzymes in Cardenolide-accumulating leaves of Digitalis lanata but also in Cardenolide-free permanent cell suspension cultures initiated from D. lanata leaf tissue. Enzyme activities were determined and quantified by HPLC and GC-MS methods. Expression of the respective genes, namely AY585867.1 (P5betaR gene) and DQ466890.1 (3beta-HSD gene), was made evident by real-time polymerase chain reaction (qPCR) analysis. We demonstrate for the first time that the P5betaR gene, encoding an enzyme described as a key enzyme in Cardenolide biosynthesis, is also expressed in Cardenolide-free tissues of Cardenolide-containing plants.
Anurag A. Agrawal - One of the best experts on this subject based on the ideXlab platform.
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less is more a mutation in the chemical defense pathway of erysimum cheiranthoides brassicaceae reduces total Cardenolide abundance but increases resistance to insect herbivores
bioRxiv, 2020Co-Authors: Mahdieh Mirzaei, Anurag A. Agrawal, Tobias Zuest, Amy P Hastings, Georg JanderAbstract:Many plants produce structurally related defensive metabolites with the same target sites in insect herbivores. Two possible drivers of this chemical diversity are: (i) interacting effects of structurally related compounds increase resistance against individual herbivores, and (ii) variants of the same chemical structures differentially affect diverse herbivore species or feeding guilds. Erysimum cheiranthoides L (Brassicaceae; wormseed wallflower) produces abundant and diverse Cardenolide toxins, which are derived from digitoxigenin, cannogenol, and strophanthidin, all of which inhibit Na+/K+-ATPases in animal cells. Here we describe an E. cheiranthoides mutant with 66% lower Cardenolide content, resulting from greatly decreased cannogenol- and strophanthidin-derived Cardenolides, partially compensated for by increases in digitoxigenin-derived Cardenolides. This compositional change created a more even Cardenolide distribution, decreased the average Cardenolide polarity, but did not impact glucosinolates, a different class of chemical defenses. Growth of generalist herbivores from two feeding guilds, Myzus persicae Sulzer (Hemiptera: Aphididae; green peach aphid) and Trichoplusia ni Hubner (Lepidoptera: Noctuidae; cabbage looper), was decreased on the mutant line compared to wildtype. Both herbivores accumulated Cardenolides in proportion to plant content, with T. ni accumulating higher total concentrations than M. persicae. Helveticoside, an abundant Cardenolide in E. cheiranthoides, was absent in M. persicae, suggesting that this compound is not present in the phloem. Our results support the hypothesis that Cardenolide diversity protects plants against different herbivores, with digitoxigenin-derived compounds providing better protection against insects like M. persicae and T. ni, whereas cannogenol and strophanthidin provide better protection against other herbivores of E. cheiranthoides. FundingThis research was funded by US National Science Foundation awards 1907491 to AAA and 1645256 to GJ and AAA, Swiss National Science Foundation grant PZ00P3-161472 to TZ, and a Triad Foundation grant to GJ.
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Cardenolide Intake, Sequestration, and Excretion by the Monarch Butterfly along Gradients of Plant Toxicity and Larval Ontogeny
Journal of Chemical Ecology, 2019Co-Authors: Patricia L. Jones, Georg Petschenka, Lara Flacht, Anurag A. AgrawalAbstract:Monarch butterflies, Danaus plexippus, migrate long distances over which they encounter host plants that vary broadly in toxic Cardenolides. Remarkably little is understood about the mechanisms of sequestration in Lepidoptera that lay eggs on host plants ranging in such toxins. Using closely-related milkweed host plants that differ more than ten-fold in Cardenolide concentrations, we mechanistically address the intake, sequestration, and excretion of Cardenolides by monarchs. We show that on high Cardenolide plant species, adult butterflies saturate in Cardenolides, resulting in lower concentrations than in leaves, while on low Cardenolide plants, butterflies concentrate toxins. Butterflies appear to focus their sequestration on particular compounds, as the diversity of Cardenolides is highest in plant leaves, lower in frass, and least in adult butterflies. Among the variety of Cardenolides produced by the plant, sequestered compounds may be less toxic to the butterflies themselves, as they are more polar on average than those in leaves. In accordance with this, results from an in vitro assay based on inhibition of Na^+/K^+ ATPase (the physiological target of Cardenolides) showed that on two milkweed species, including the high Cardenolide A. perennis , extracts from butterflies have lower inhibitory effects than leaves when standardized by Cardenolide concentration, indicating selective sequestration of less toxic compounds from these host plants. To understand how ontogeny shapes sequestration, we examined Cardenolide concentrations in caterpillar body tissues and hemolymph over the course of development. Caterpillars sequestered higher concentrations of Cardenolides as early instars than as late instars, but within the fifth instar, concentration increased with body mass. Although it appears that large amounts of sequestration occurs in early instars, a host switching experiment revealed that caterpillars can compensate for feeding on low Cardenolide host plants with substantial sequestration in the fifth instar. We highlight commonalities and striking differences in the mechanisms of sequestration depending on host plant chemistry and developmental stage, which have important implications for monarch defense.
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multidrug transporters and organic anion transporting polypeptides protect insects against the toxic effects of Cardenolides
Insect Biochemistry and Molecular Biology, 2017Co-Authors: Simon C Groen, Susanne Dobler, Anurag A. Agrawal, Erika R Laplante, Nicolas M Alexandre, Noah K WhitemanAbstract:In the struggle against dietary toxins, insects are known to employ target site insensitivity, metabolic detoxification, and transporters that shunt away toxins. Specialized insects across six taxonomic orders feeding on Cardenolide-containing plants have convergently evolved target site insensitivity via specific amino acid substitutions in the Na/K-ATPase. Nonetheless, in vitro pharmacological experiments have suggested a role for multidrug transporters (Mdrs) and organic anion transporting polypeptides (Oatps), which may provide a basal level of protection in both specialized and non-adapted insects. Because the genes coding for these proteins are evolutionarily conserved and in vivo genetic evidence in support of this hypothesis is lacking, here we used wildtype and mutant Drosophila melanogaster (Drosophila) in capillary feeder (CAFE) assays to quantify toxicity of three chemically diverse, medically relevant Cardenolides. We examined multiple components of fitness, including mortality, longevity, and LD50, and found that, while the three Cardenolides each stimulated feeding (i.e., no deterrence to the toxin), all decreased lifespan, with the most apolar Cardenolide having the lowest LD50 value. Flies showed a clear non-monotonic dose response and experienced high levels of toxicity at the Cardenolide concentration found in plants. At this concentration, both Mdr and Oatp knockout mutant flies died more rapidly than wildtype flies, and the mutants also experienced more adverse neurological effects on high-Cardenolide-level diets. Our study further establishes Drosophila as a model for the study of Cardenolide pharmacology and solidifies support for the hypothesis that multidrug and organic anion transporters are key players in insect protection against dietary Cardenolides.
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Milkweed butterfly resistance to plant toxins is linked to sequestration, not coping with a toxic diet.
Proceedings. Biological sciences, 2015Co-Authors: Georg Petschenka, Anurag A. AgrawalAbstract:Insect resistance to plant toxins is widely assumed to have evolved in response to using defended plants as a dietary resource. We tested this hypothesis in the milkweed butterflies (Danaini) which have progressively evolved higher levels of resistance to Cardenolide toxins based on amino acid substitutions of their cellular sodium-potassium pump (Na(+)/K(+)-ATPase). Using chemical, physiological and caterpillar growth assays on diverse milkweeds (Asclepias spp.) and isolated Cardenolides, we show that resistant Na(+)/K(+)-ATPases are not necessary to cope with dietary Cardenolides. By contrast, sequestration of Cardenolides in the body (as a defence against predators) is associated with the three levels of Na(+)/K(+)-ATPase resistance. To estimate the potential physiological burden of Cardenolide sequestration without Na(+)/K(+)-ATPase adaptations, we applied haemolymph of sequestering species on isolated Na(+)/K(+)-ATPase of sequestering and non-sequestering species. Haemolymph Cardenolides dramatically impair non-adapted Na(+)/K(+)-ATPase, but had systematically reduced effects on Na(+)/K(+)-ATPase of sequestering species. Our data indicate that major adaptations to plant toxins may be evolutionarily linked to sequestration, and may not necessarily be a means to eat toxic plants. Na(+)/K(+)-ATPase adaptations thus were a potential mechanism through which predators spurred the coevolutionary arms race between plants and insects.
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Population growth and sequestration of plant toxins along a gradient of specialization in four aphid species on the common milkweed Asclepias syriaca
Functional Ecology, 2015Co-Authors: Tobias Züst, Anurag A. AgrawalAbstract:Summary 1. Dietary specialization in insect herbivores has long been hypothesized to predict tolerance of plant defences, with more specialized herbivores being highly tolerant of and sometimes sequestering plant secondary compounds. Plant variation in secondary compounds should thus play an important and predictable role in shaping the performance and distribution of insect communities. 2. We compared the performance of four naturally co-occurring aphid species on twenty genotypes of the common milkweed Asclepias syriaca. Genotypes of milkweed consistently differed in functional traits, including concentrations of toxic Cardenolides, while the diet breadths of the four aphids ranged from broadly generalized to monophagous. 3. The two more generalized species had the highest population growth rate overall, while growth rates decreased with increasing specialization. In contrast, honeydew exudation as a measure of phloem consumption increased with specialization; thus, resource-use efficiency was lower in specialist aphids. The two more generalized aphids grew best on genotypes with the highest plant growth rate (as an approximation for resource availability), while specialist aphids were not affected by plant growth. 4. All four species contained apolar Cardenolides in their bodies and excreted polar Cardenolides, but only the most specialized aphid Myzocallis asclepiadis was negatively affected by increasing Cardenolide concentrations of the host plant. Sequestration of Cardenolides increased with diet specialization, with M. asclepiadis accumulating twice as much as any other species, perhaps explaining its susceptibility to plant Cardenolides. 5. Heritable plant traits differentially impacted co-occurring insect herbivores within the same guild. Generalist aphids were susceptible to variation in plant vigour but not defensive compounds. Increased host specialization resulted in lower resource-use efficiency, increased phloem throughput and ultimately higher Cardenolide sequestration. Variation in these traits is thus likely to determine the relative distribution of generalist and specialist herbivores on plants in natural communities.
Susanne Dobler - One of the best experts on this subject based on the ideXlab platform.
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the function and evolutionary significance of a triplicated na k atpase gene in a toxin specialized insect
BMC Evolutionary Biology, 2017Co-Authors: Jennifer N Lohr, Safaa Dalla, Fee Meinzer, Renja Romeyglusing, Susanne DoblerAbstract:The Na,K-ATPase is a vital animal cell-membrane protein that maintains the cell’s resting potential, among other functions. Cardenolides, a group of potent plant toxins, bind to and inhibit this pump. The gene encoding the α-subunit of the pump has undergone duplication events in some insect species known to feed on plants containing Cardenolides. Here we test the function of these duplicated gene copies in the Cardenolide-adapted milkweed bug, Oncopeltus fasciatus, which has three known copies of the gene: α1A, α1B and α1C. Using RT-qPCR analyses we demonstrate that the α1C is highly expressed in neural tissue, where the pump is generally thought to be most important for neuron excitability. With the use of in vivo RNAi in adult bugs we found that α1C knockdowns suffered high mortality, where as α1A and α1B did not, supporting that α1C is most important for effective ion pumping. Next we show a role for α1A and α1B in the handling of Cardenolides: expression results find that both copies are primarily expressed in the Malpighian tubules, the primary insect organ responsible for excretion, and when we injected either α1A or α1B knockdowns with Cardenolides this proved fatal (whereas not in controls). These results show that the Na,K-ATPα gene-copies have taken on diverse functions. Having multiple copies of this gene appears to have allowed the newly arisen duplicates to specialize on resistance to Cardenolides, whereas the ancestral copy of the pump remains comparatively sensitive, but acts as a more efficient ion carrier. Interestingly both the α1A and α1B were required for Cardenolide handling, suggesting that these two copies have separate and vital functions. Gene duplications of the Na,K-ATPase thus represent an excellent example of subfunctionalization in response to a new environmental challenge.
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multidrug transporters and organic anion transporting polypeptides protect insects against the toxic effects of Cardenolides
Insect Biochemistry and Molecular Biology, 2017Co-Authors: Simon C Groen, Susanne Dobler, Anurag A. Agrawal, Erika R Laplante, Nicolas M Alexandre, Noah K WhitemanAbstract:In the struggle against dietary toxins, insects are known to employ target site insensitivity, metabolic detoxification, and transporters that shunt away toxins. Specialized insects across six taxonomic orders feeding on Cardenolide-containing plants have convergently evolved target site insensitivity via specific amino acid substitutions in the Na/K-ATPase. Nonetheless, in vitro pharmacological experiments have suggested a role for multidrug transporters (Mdrs) and organic anion transporting polypeptides (Oatps), which may provide a basal level of protection in both specialized and non-adapted insects. Because the genes coding for these proteins are evolutionarily conserved and in vivo genetic evidence in support of this hypothesis is lacking, here we used wildtype and mutant Drosophila melanogaster (Drosophila) in capillary feeder (CAFE) assays to quantify toxicity of three chemically diverse, medically relevant Cardenolides. We examined multiple components of fitness, including mortality, longevity, and LD50, and found that, while the three Cardenolides each stimulated feeding (i.e., no deterrence to the toxin), all decreased lifespan, with the most apolar Cardenolide having the lowest LD50 value. Flies showed a clear non-monotonic dose response and experienced high levels of toxicity at the Cardenolide concentration found in plants. At this concentration, both Mdr and Oatp knockout mutant flies died more rapidly than wildtype flies, and the mutants also experienced more adverse neurological effects on high-Cardenolide-level diets. Our study further establishes Drosophila as a model for the study of Cardenolide pharmacology and solidifies support for the hypothesis that multidrug and organic anion transporters are key players in insect protection against dietary Cardenolides.
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The function and evolutionary significance of a triplicated Na,K-ATPase gene in a toxin-specialized insect
BMC, 2017Co-Authors: Jennifer N Lohr, Safaa Dalla, Fee Meinzer, Renja Romey-glüsing, Susanne DoblerAbstract:Abstract Background The Na,K-ATPase is a vital animal cell-membrane protein that maintains the cell’s resting potential, among other functions. Cardenolides, a group of potent plant toxins, bind to and inhibit this pump. The gene encoding the α-subunit of the pump has undergone duplication events in some insect species known to feed on plants containing Cardenolides. Here we test the function of these duplicated gene copies in the Cardenolide-adapted milkweed bug, Oncopeltus fasciatus, which has three known copies of the gene: α1A, α1B and α1C. Results Using RT-qPCR analyses we demonstrate that the α1C is highly expressed in neural tissue, where the pump is generally thought to be most important for neuron excitability. With the use of in vivo RNAi in adult bugs we found that α1C knockdowns suffered high mortality, where as α1A and α1B did not, supporting that α1C is most important for effective ion pumping. Next we show a role for α1A and α1B in the handling of Cardenolides: expression results find that both copies are primarily expressed in the Malpighian tubules, the primary insect organ responsible for excretion, and when we injected either α1A or α1B knockdowns with Cardenolides this proved fatal (whereas not in controls). Conclusions These results show that the Na,K-ATPα gene-copies have taken on diverse functions. Having multiple copies of this gene appears to have allowed the newly arisen duplicates to specialize on resistance to Cardenolides, whereas the ancestral copy of the pump remains comparatively sensitive, but acts as a more efficient ion carrier. Interestingly both the α1A and α1B were required for Cardenolide handling, suggesting that these two copies have separate and vital functions. Gene duplications of the Na,K-ATPase thus represent an excellent example of subfunctionalization in response to a new environmental challenge
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na k atpase resistance and Cardenolide sequestration basal adaptations to host plant toxins in the milkweed bugs hemiptera lygaeidae lygaeinae
Proceedings of The Royal Society B: Biological Sciences, 2015Co-Authors: Christiane Bramer, Jürgen Deckert, Susanne Dobler, Michael Stemmer, Georg PetschenkaAbstract:Despite sequestration of toxins being a common coevolutionary response to plant defence in phytophagous insects, the macroevolution of the traits involved is largely unaddressed. Using a phylogenetic approach comprising species from four continents, we analysed the ability to sequester toxic Cardenolides in the hemipteran subfamily Lygaeinae, which is widely associated with Cardenolide-producing Apocynaceae. In addition, we analysed Cardenolide resistance of their Na+/K+-ATPases, the molecular target of Cardenolides. Our data indicate that Cardenolide sequestration and Cardenolide-resistant Na+/K+-ATPase are basal adaptations in the Lygaeinae. In two species that shifted to non-apocynaceous hosts, the ability to sequester was secondarily reduced, yet Na+/K+-ATPase resistance was maintained. We suggest that both traits evolved together and represent major coevolutionary adaptations responsible for the evolutionary success of lygaeine bugs. Moreover, specialization on Cardenolides was not an evolutionary dead end, but enabled this insect lineage to host shift to Cardenolide-producing plants from distantly related families.
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amino acid substitutions of na k atpase conferring decreased sensitivity to Cardenolides in insects compared to mammals
Insect Biochemistry and Molecular Biology, 2013Co-Authors: Safaa Dalla, Herman G P Swarts, Jan B Koenderink, Susanne DoblerAbstract:Mutagenesis analyses and a recent crystal structure of the mammalian Na,K-ATPase have identified amino acids which are responsible for high affinity binding of Cardenolides (such as ouabain) which at higher doses block the enzyme in the phosphorylated state. Genetic analysis of the Na,K-ATPase of insects adapted to Cardenolides in their food plants revealed that some species possess substitutions which confer strongly increased resistance to ouabain in the mammalian enzyme such as the substitution T797A or combined substitutions at positions 111 and 122. To test for the effect of these mutations against the background of insect Na,K-ATPase, we here expressed the ouabain sensitive Na,K-ATPase alpha-subunit of Drosophila melanogaster together with the beta-subunit Nrv3 in baculovirus-infected Sf9 cells and introduced the substitutions N122H, T797A, Q111T-N122H, Q111V-N122H, all of which have been observed in Cardenolide-adapted insects. While all constructs showed similar expression levels, ouabain affinity of mutated Na,K-ATPases was reduced compared to the wild-type fly enzyme. Ouabain sensitivity of the ATPase activity in inhibition assays was significantly decreased by all mutations, yet whereas the IC50 for the single mutations of N122H (61.0 muM) or T797A (63.3 muM) was increased roughly 250-fold relative to the wild-type (0.24 muM), the double mutations of Q111V-N122H (IC50 550 muM) and Q111T-N122H (IC50 583 muM) proved to be still more effective yielding a 2.250-fold increased resistance to ouabain. The double mutations identified in Cardenolide-adapted insects are more effective in reducing ouabain sensitivity of the enzyme than those found naturally in the rat Na,K-ATPase (Q111R-N122D) or in mutagenesis screens of the mammalian enzyme. Obviously, the intense selection pressure on Cardenolide exposed insects has resulted in very efficient substitutions that decrease Cardenolide sensitivity extremely.
Georg Petschenka - One of the best experts on this subject based on the ideXlab platform.
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Cardenolide Intake, Sequestration, and Excretion by the Monarch Butterfly along Gradients of Plant Toxicity and Larval Ontogeny
Journal of Chemical Ecology, 2019Co-Authors: Patricia L. Jones, Georg Petschenka, Lara Flacht, Anurag A. AgrawalAbstract:Monarch butterflies, Danaus plexippus, migrate long distances over which they encounter host plants that vary broadly in toxic Cardenolides. Remarkably little is understood about the mechanisms of sequestration in Lepidoptera that lay eggs on host plants ranging in such toxins. Using closely-related milkweed host plants that differ more than ten-fold in Cardenolide concentrations, we mechanistically address the intake, sequestration, and excretion of Cardenolides by monarchs. We show that on high Cardenolide plant species, adult butterflies saturate in Cardenolides, resulting in lower concentrations than in leaves, while on low Cardenolide plants, butterflies concentrate toxins. Butterflies appear to focus their sequestration on particular compounds, as the diversity of Cardenolides is highest in plant leaves, lower in frass, and least in adult butterflies. Among the variety of Cardenolides produced by the plant, sequestered compounds may be less toxic to the butterflies themselves, as they are more polar on average than those in leaves. In accordance with this, results from an in vitro assay based on inhibition of Na^+/K^+ ATPase (the physiological target of Cardenolides) showed that on two milkweed species, including the high Cardenolide A. perennis , extracts from butterflies have lower inhibitory effects than leaves when standardized by Cardenolide concentration, indicating selective sequestration of less toxic compounds from these host plants. To understand how ontogeny shapes sequestration, we examined Cardenolide concentrations in caterpillar body tissues and hemolymph over the course of development. Caterpillars sequestered higher concentrations of Cardenolides as early instars than as late instars, but within the fifth instar, concentration increased with body mass. Although it appears that large amounts of sequestration occurs in early instars, a host switching experiment revealed that caterpillars can compensate for feeding on low Cardenolide host plants with substantial sequestration in the fifth instar. We highlight commonalities and striking differences in the mechanisms of sequestration depending on host plant chemistry and developmental stage, which have important implications for monarch defense.
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Milkweed butterfly resistance to plant toxins is linked to sequestration, not coping with a toxic diet.
Proceedings. Biological sciences, 2015Co-Authors: Georg Petschenka, Anurag A. AgrawalAbstract:Insect resistance to plant toxins is widely assumed to have evolved in response to using defended plants as a dietary resource. We tested this hypothesis in the milkweed butterflies (Danaini) which have progressively evolved higher levels of resistance to Cardenolide toxins based on amino acid substitutions of their cellular sodium-potassium pump (Na(+)/K(+)-ATPase). Using chemical, physiological and caterpillar growth assays on diverse milkweeds (Asclepias spp.) and isolated Cardenolides, we show that resistant Na(+)/K(+)-ATPases are not necessary to cope with dietary Cardenolides. By contrast, sequestration of Cardenolides in the body (as a defence against predators) is associated with the three levels of Na(+)/K(+)-ATPase resistance. To estimate the potential physiological burden of Cardenolide sequestration without Na(+)/K(+)-ATPase adaptations, we applied haemolymph of sequestering species on isolated Na(+)/K(+)-ATPase of sequestering and non-sequestering species. Haemolymph Cardenolides dramatically impair non-adapted Na(+)/K(+)-ATPase, but had systematically reduced effects on Na(+)/K(+)-ATPase of sequestering species. Our data indicate that major adaptations to plant toxins may be evolutionarily linked to sequestration, and may not necessarily be a means to eat toxic plants. Na(+)/K(+)-ATPase adaptations thus were a potential mechanism through which predators spurred the coevolutionary arms race between plants and insects.
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na k atpase resistance and Cardenolide sequestration basal adaptations to host plant toxins in the milkweed bugs hemiptera lygaeidae lygaeinae
Proceedings of The Royal Society B: Biological Sciences, 2015Co-Authors: Christiane Bramer, Jürgen Deckert, Susanne Dobler, Michael Stemmer, Georg PetschenkaAbstract:Despite sequestration of toxins being a common coevolutionary response to plant defence in phytophagous insects, the macroevolution of the traits involved is largely unaddressed. Using a phylogenetic approach comprising species from four continents, we analysed the ability to sequester toxic Cardenolides in the hemipteran subfamily Lygaeinae, which is widely associated with Cardenolide-producing Apocynaceae. In addition, we analysed Cardenolide resistance of their Na+/K+-ATPases, the molecular target of Cardenolides. Our data indicate that Cardenolide sequestration and Cardenolide-resistant Na+/K+-ATPase are basal adaptations in the Lygaeinae. In two species that shifted to non-apocynaceous hosts, the ability to sequester was secondarily reduced, yet Na+/K+-ATPase resistance was maintained. We suggest that both traits evolved together and represent major coevolutionary adaptations responsible for the evolutionary success of lygaeine bugs. Moreover, specialization on Cardenolides was not an evolutionary dead end, but enabled this insect lineage to host shift to Cardenolide-producing plants from distantly related families.
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stepwise evolution of resistance to toxic Cardenolides via genetic substitutions in the na k atpase of milkweed butterflies lepidoptera danaini
Evolution, 2013Co-Authors: Georg Petschenka, Michael Boppré, Vera Wagschal, Steffi Fandrich, Nils Sander, Susanne DoblerAbstract:Despite the monarch butterfly (Danaus plexippus) being famous for its adaptations to the defensive traits of its milkweed host plants, little is known about the macroevolution of these traits. Unlike most other animal species, monarchs are largely insensitive to Cardenolides, because their target site, the sodium pump (Na(+)/K(+) -ATPase), has evolved amino acid substitutions that reduce Cardenolide binding (so-called target site insensitivity, TSI). Because many, but not all, species of milkweed butterflies (Danaini) are associated with Cardenolide-containing host plants, we analyzed 16 species, representing all phylogenetic lineages of milkweed butterflies, for the occurrence of TSI by sequence analyses of the Na(+)/K(+) -ATPase gene and by enzymatic assays with extracted Na(+)/K(+) -ATPase. Here we report that sensitivity to Cardenolides was reduced in a stepwise manner during the macroevolution of milkweed butterflies. Strikingly, not all Danaini typically consuming Cardenolides showed TSI, but rather TSI was more strongly associated with sequestration of toxic Cardenolides. Thus, the interplay between bottom-up selection by plant compounds and top-down selection by natural enemies can explain the evolutionary sequence of adaptations to these toxins.
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functional evidence for physiological mechanisms to circumvent neurotoxicity of Cardenolides in an adapted and a non adapted hawk moth species
Proceedings of The Royal Society B: Biological Sciences, 2013Co-Authors: Georg Petschenka, Christian Pick, Vera Wagschal, Susanne DoblerAbstract:Because Cardenolides specifically inhibit the Na+K+-ATPase, insects feeding on Cardenolide-containing plants need to circumvent this toxic effect. Some insects such as the monarch butterfly rely on target site insensitivity, yet other Cardenolide-adapted lepidopterans such as the oleander hawk-moth, Daphnis nerii , possess highly sensitive Na+K+-ATPases. Nevertheless, larvae of this species and the related Manduca sexta are insensitive to injected Cardenolides. By radioactive-binding assays with nerve cords of both species, we demonstrate that the perineurium surrounding the nervous tissue functions as a diffusion barrier for a polar Cardenolide (ouabain). By contrast, for non-polar Cardenolides such as digoxin an active efflux carrier limits the access to the nerve cord. This barrier can be abolished by metabolic inhibitors and by verapamil, a specific inhibitor of P-glycoproteins (PGPs). This supports that a PGP-like transporter is involved in the active Cardenolide-barrier of the perineurium. Tissue specific RT-PCR demonstrated expression of three PGP-like genes in hornworm nerve cords, and immunohistochemistry further corroborated PGP expression in the perineurium. Our results thus suggest that the lepidopteran perineurium serves as a diffusion barrier for polar Cardenolides and provides an active barrier for non-polar Cardenolides. This may explain the high in vivo resistance to Cardenolides observed in some lepidopteran larvae, despite their highly sensitive Na+K+-ATPases.
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acropetal and basipetal Cardenolide transport in erysimum cheiranthoides wormseed wallflower
Phytochemistry, 2021Co-Authors: Martin L Alani, Mahdieh Mirzaei, Pavan Kumar, Gordon C Younkin, Georg JanderAbstract:Abstract Plant specialized metabolites are often subject to within-plant transport and have tissue-specific distribution patterns. Among plants in the Brassicaceae, the genus Erysimum is unique in producing not only glucosinolates but also Cardenolides. Ten Cardenolides were detected with varying abundance in different tissues of Erysimum cheiranthoides L (Brassicaceae; wormseed wallflower). As is predicted by the optimal defense theory, Cardenolides were most abundant in young leaves and reproductive tissues. The lowest concentrations were observed in senescing leaves and roots. Crosses between wildtype E. cheiranthoides and a mutant line with an altered Cardenolide profile showed that the seed Cardenolide phenotype is determined entirely by the maternal genotype. Prior to the development of the first true leaves, seedling cotyledons also had the maternal Cardenolide profile. Hypocotyl grafting experiments showed that the root Cardenolide profile is determined entirely by the aboveground plant genotype. In further grafting experiments, there was no evidence of Cardenolide transport into the leaves, but a mixed Cardenolide profile was observed in the stems and inflorescences of plants that had been grafted at vegetative and flowering growth stages, respectively. Together, these results indicate that E. cheiranthoides leaves are a site of Cardenolide biosynthesis.
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acropetal and basipetal Cardenolide transport in erysimum cheiranthoides wormseed wallflower
bioRxiv, 2021Co-Authors: Martin L Alani, Mahdieh Mirzaei, Pavan Kumar, Gordon C Younkin, Georg JanderAbstract:Plant specialized metabolites are often subject to within-plant transport and have tissue-specific distribution patterns. Among plants in the Brassicaceae, the genus Erysimum is unique in producing not only glucosinolates but also Cardenolides as defense against insect herbivory. Ten Cardenolides were detected with varying abundance in different tissues of Erysimum cheiranthoides (wormseed wallflower). As is predicted by the optimal defense theory, Cardenolides were most abundant in young leaves and reproductive tissues. The lowest concentrations were observed in senescing leaves and roots. Crosses between wildtype E. cheiranthoides and a mutant line with an altered Cardenolide profile showed that the seed Cardenolide phenotype is determined entirely by the maternal genotype. Prior to the development of the first true leaves, seedling cotyledons also had the maternal Cardenolide profile. Hypocotyl grafting experiments showed that the root Cardenolide profile is determined entirely by the aboveground plant genotype. In further grafting experiments, there was no evidence of Cardenolide transport into the leaves, but a mixed Cardenolide profile was observed in the stems and inflorescences of plants that had been grafted at vegetative and flowering growth stages, respectively. Together, these results indicate that E. cheiranthoides leaves are a likely site of Cardenolide biosynthesis and therefore also likely the plant tissue that is most likely to be expressing the relevant biosynthetic genes.
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acropetal and basipetal Cardenolide transport in erysimum cheiranthoides wormseed wallflower
bioRxiv, 2021Co-Authors: Martin L Alani, Mahdieh Mirzaei, Pavan Kumar, Gordon C Younkin, Georg JanderAbstract:Abstract Plant specialized metabolites are often subject to within-plant transport and have tissue-specific distribution patterns. Among plants in the Brassicaceae, the genus Erysimum is unique in producing not only glucosinolates but also Cardenolides as defense against insect herbivory. Ten Cardenolides were detected with varying abundance in different tissues of Erysimum cheiranthoides (wormseed wallflower). As is predicted by the optimal defense theory, Cardenolides were most abundant in young leaves and reproductive tissues. The lowest concentrations were observed in senescing leaves and roots. Crosses between wildtype E. cheiranthoides and a mutant line with an altered Cardenolide profile showed that the seed Cardenolide phenotype is determined entirely by the maternal genotype. Prior to the development of the first true leaves, seedling cotyledons also had the maternal Cardenolide profile. Hypocotyl grafting experiments showed that the root Cardenolide profile is determined entirely by the aboveground plant genotype. In further grafting experiments, there was no evidence of Cardenolide transport into the leaves, but a mixed Cardenolide profile was observed in the stems and inflorescences of plants that had been grafted at vegetative and flowering growth stages, respectively. Together, these results indicate that E. cheiranthoides leaves are a site of Cardenolide biosynthesis and therefore also the plant tissue that is most likely to be expressing the relevant biosynthetic genes.
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less is more a mutation in the chemical defense pathway of erysimum cheiranthoides brassicaceae reduces total Cardenolide abundance but increases resistance to insect herbivores
bioRxiv, 2020Co-Authors: Mahdieh Mirzaei, Anurag A. Agrawal, Tobias Zuest, Amy P Hastings, Georg JanderAbstract:Many plants produce structurally related defensive metabolites with the same target sites in insect herbivores. Two possible drivers of this chemical diversity are: (i) interacting effects of structurally related compounds increase resistance against individual herbivores, and (ii) variants of the same chemical structures differentially affect diverse herbivore species or feeding guilds. Erysimum cheiranthoides L (Brassicaceae; wormseed wallflower) produces abundant and diverse Cardenolide toxins, which are derived from digitoxigenin, cannogenol, and strophanthidin, all of which inhibit Na+/K+-ATPases in animal cells. Here we describe an E. cheiranthoides mutant with 66% lower Cardenolide content, resulting from greatly decreased cannogenol- and strophanthidin-derived Cardenolides, partially compensated for by increases in digitoxigenin-derived Cardenolides. This compositional change created a more even Cardenolide distribution, decreased the average Cardenolide polarity, but did not impact glucosinolates, a different class of chemical defenses. Growth of generalist herbivores from two feeding guilds, Myzus persicae Sulzer (Hemiptera: Aphididae; green peach aphid) and Trichoplusia ni Hubner (Lepidoptera: Noctuidae; cabbage looper), was decreased on the mutant line compared to wildtype. Both herbivores accumulated Cardenolides in proportion to plant content, with T. ni accumulating higher total concentrations than M. persicae. Helveticoside, an abundant Cardenolide in E. cheiranthoides, was absent in M. persicae, suggesting that this compound is not present in the phloem. Our results support the hypothesis that Cardenolide diversity protects plants against different herbivores, with digitoxigenin-derived compounds providing better protection against insects like M. persicae and T. ni, whereas cannogenol and strophanthidin provide better protection against other herbivores of E. cheiranthoides. FundingThis research was funded by US National Science Foundation awards 1907491 to AAA and 1645256 to GJ and AAA, Swiss National Science Foundation grant PZ00P3-161472 to TZ, and a Triad Foundation grant to GJ.