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May R Berenbaum - One of the best experts on this subject based on the ideXlab platform.
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substrate specificity of cytochrome p450 mediated detoxification as an evolutionary strategy for specialization on furanocoumarin containing hostplants cyp6ae89 in parsnip webworms
Insect Molecular Biology, 2020Co-Authors: Bernarda Calla, Mary A Schuler, Charles A E Dean, May R BerenbaumAbstract:The parsnip webworm, Depressaria pastinacella, is restricted to two hostplant genera containing six structurally diverse Furanocoumarins. Of these, imperatorin is detoxified by a specialized cytochrome P450, CYP6AB3. A previous whole-larva transcriptome analysis confirmed the presence of nine transcripts that belong to the CYP6AE subfamily. Here, by examining midgut-specific gene expression patterns we determined that CYP6AE89 transcripts were highly expressed and furanocoumarin-inducible. Computer docking and energy-minimization of a CYP6AE89 model with all six Furanocoumarins showed that 5-methoxylated bergapten and 8-methoxylated xanthotoxin had the smallest distances from the heme to the proton-donor residue in the catalytic I-helix, and that the 5,8-dimethoxylated isopimpinellin and bergapten had the smallest energy-minimized distance from the heme oxygen to the furan ring double bond. To evaluate this prediction, we expressed the CYP6AE89 protein in an Escherichia coli system, and used it to detect high catalytic activity against the two mono-methoxylated linear Furanocoumarins - bergapten and xanthotoxin - and weak activity against isopimpinellin. Thus, CYP6AE89, like CYP6AB3, is probably specialized for detoxifying only a subset of hostplant Furanocoumarins. A maximum-likelihood tree built with six representative lepidopterans with manually annotated cytochrome P450s shows that CYP6AE89 may have evolved much faster than the other CYP6AE proteins, possibly indicative of host selection pressure.
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Accelerated Development and Toxin Tolerance of the Navel Orangeworm Amyelois transitella (Lepidoptera: Pyralidae) in the Presence of Aspergillus flavus
Journal of Chemical Ecology, 2018Co-Authors: Daniel S Bush, Joel P. Siegel, May R BerenbaumAbstract:The navel orangeworm ( Amyelois transitella ) and the fungus Aspergillus flavus constitute a facultative mutualism and pest complex in tree nut and fruit orchards in California. The possibility exists that the broad detoxification capabilities of A. flavus benefit its insect associate by metabolizing toxicants, including hostplant phytochemicals and pesticides. We examined this hypothesis by conducting laboratory bioassays to assess growth rates and survivorship of pyrethroid-resistant (R347) and susceptible (CPQ) larval strains on potato dextrose agar diet containing almond meal with and without two Furanocoumarins, xanthotoxin and bergapten, found in several hostplants, and with and without two insecticides, bifenthrin and spinetoram, used in almond and pistachio orchards. Additionally, fungi were incubated in liquid diets containing the test chemicals, and extracts of these diets were added to almond potato dextrose agar (PDA) diets and fed to larvae to evaluate the ability of the fungus to metabolize these chemicals. Larvae consuming furanocoumarin-containing diet experienced higher mortality than individuals on unamended diets, but adding A. flavus resulted in up to 61.7% greater survival. Aspergillus flavus in the diet increased development rate > two-fold when Furanocoumarins were present, demonstrating fungal enhancement of diet quality. Adding extracts of liquid diets containing xanthotoxin and fungus decreased mortality compared to xanthotoxin alone. On diets containing bifenthrin and spinetoram, however, mortality increased. These results support the hypothesis that A. flavus enhances navel orangeworm performance and contributes to detoxification of xenobiotics. Among practical implications of our findings, this mutualistic association should be considered in designing chemical management strategies for these pests.
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cyp6b1 and cyp6b3 of the black swallowtail papilio polyxenes adaptive evolution through subfunctionalization
Molecular Biology and Evolution, 2006Co-Authors: Sanjeewa G Rupasinghe, May R Berenbaum, Mary A SchulerAbstract:Gene duplication provides essential material for functional divergence of proteins and hence allows organisms to adapt to changing environments. Following duplication events, redundant paralogs may undergo different evolutionary paths via processes known as nonfunctionalization, neofunctionalization, or subfunctionalization. Studies of adaptive evolution at the molecular level have progressed rapidly by computationally analyzing nucleotide substitution patterns but such studies are limited by the absence of information relating to alterations of function of the encoded enzymes. In this respect, evolution of the Papilio polyxenes cytochrome P450 monooxygenases (P450s) responsible for the adaptation of this insect to furanocoumarin-containing host plants provides an excellent model for elucidating the evolutionary fate of duplicated genes. Evidence from sequence and functional analysis in combination with molecular modeling indicates that the paralogous CYP6BI and CYP6B3 genes in P. polyxenes have probably evolved via subfunctionalization after the duplication event by which they arose. Both enzymes have been under independent purifying selection as evidenced by the low dN/dS ratio in both the coding region and substrate recognition sites. Both enzymes have maintained their ability to metabolize linear and angular Furanocoumarins albeit at different efficiencies. Comparisons of molecular models developed for the CYP6B3 and CYP6B1 proteins highlight differences in their binding modes that account for their different activities toward linear and angular Furanocoumarins. That P. polyxenes maintains these 2 furanocoumarin-metabolizing loci with somewhat different activities and expression patterns provides this species with the potential to acquire P450s with novel functions while maintaining those most critical to its exclusive feeding on its current range of host plants.
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Lutein Sequestration and Furanocoumarin Metabolism in Parsnip Webworms Under Different Ultraviolet Light Regimes in the Montane West
Journal of Chemical Ecology, 2006Co-Authors: Mark J. Carroll, May R BerenbaumAbstract:Both biotic and abiotic selection pressures can contribute to geographic variation in allelochemical production in plants. We examined furanocoumarin production in western North American populations of Heracleum lanatum and Pastinaca sativa that, at different latitudes and altitudes, experience different ultraviolet (UV) light regimes. Total Furanocoumarins and linear Furanocoumarins of fruits were negatively correlated with UV irradiance, whereas amounts of angular Furanocoumarins, which are generally less phototoxic, were not. Another factor potentially influencing furanocoumarin production is the presence of the parsnip webworm Depressaria pastinacella , (Lepidoptera: Oecophoridae), an herbivore that feeds on reproductive structures of both plant species. These insects sequester lutein from their host plants; this carotenoid acts to ameliorate furanocoumarin toxicity. Although the concentration of lutein in fruits did not vary with UV irradiance, lutein sequestration by sixth instars was positively correlated with UV irradiance. Webworm populations are variably infested with the polyembryonic webworm parasitoid Copidosoma sosares Walker (Hymenoptera: Encyrtidae). H. lanatum fruits from populations with webworms parasitized by C. sosares had lower concentrations of Furanocoumarins, with the exception of sphondin, than fruits from plants infested with webworms free from parasitism. Lower levels of these Furanocoumarins may reduce negative effects on the fitness of this parasitoid. In contrast with the variation in furanocoumarin content, the ability of webworms to metabolize Furanocoumarins by cytochrome P450 did not differ significantly among populations from New Mexico to Alberta.
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characterization and evolution of furanocoumarin inducible cytochrome p450s in the parsnip webworm depressaria pastinacella
Insect Molecular Biology, 2004Co-Authors: W. Li, Arthur R. Zangerl, Mary A Schuler, May R BerenbaumAbstract:: Depressaria pastinacella, the parsnip webworm, a specialist on two genera in the Apiaceae, routinely consumes plant tissues high in furanocoumarin content and is capable of rapid cytochrome P450-mediated detoxification of these compounds. In this study, four cDNAs were cloned from the larval midgut of this insect: two full-length CYP6AB3 and CYP6AE1 cDNAs are closely related to members of the furanocoumarin-metabolizing CYP6B subfamily and two partial CYP9A6 and CYP9A7 cDNAs are related to members of the CYP9A subfamily that have also been linked to the detoxification of xenobiotics. At least one of these P450s (CYP6AB3) is inducible by dietary Furanocoumarins, indicating its potential involvement in furanocoumarin metabolism. A homology model of CYP6AB3 was constructed and compared to models of CYP6B1 from the specialist species, Papilio polyxenes, and CYP6B4 from the generalist species, P. glaucus. Structural superpositioning of these models has revealed very high spatial similarity of elements, including the B helix, B'-C loop, I helix and C-terminal domain, within the catalytic sites of these proteins. Most importantly, key amino acid residues that can potentially come into contact with furanocoumarin substrates display conservation in their spatial positioning and side chain polarities. Three of these residues, Val103, Leu113 and Phe118 (numbered according to CYP6AB3), are conserved in all three of these proteins, further implicating CYP6AB3 in furanocoumarin metabolism by parsnip webworms. Characterization of these P450 cDNAs will allow for functional analyses aimed at elucidating the molecular mechanisms underlying the coevolutionary interactions between this herbivore and its principal host plant.
Mary A Schuler - One of the best experts on this subject based on the ideXlab platform.
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substrate specificity of cytochrome p450 mediated detoxification as an evolutionary strategy for specialization on furanocoumarin containing hostplants cyp6ae89 in parsnip webworms
Insect Molecular Biology, 2020Co-Authors: Bernarda Calla, Mary A Schuler, Charles A E Dean, May R BerenbaumAbstract:The parsnip webworm, Depressaria pastinacella, is restricted to two hostplant genera containing six structurally diverse Furanocoumarins. Of these, imperatorin is detoxified by a specialized cytochrome P450, CYP6AB3. A previous whole-larva transcriptome analysis confirmed the presence of nine transcripts that belong to the CYP6AE subfamily. Here, by examining midgut-specific gene expression patterns we determined that CYP6AE89 transcripts were highly expressed and furanocoumarin-inducible. Computer docking and energy-minimization of a CYP6AE89 model with all six Furanocoumarins showed that 5-methoxylated bergapten and 8-methoxylated xanthotoxin had the smallest distances from the heme to the proton-donor residue in the catalytic I-helix, and that the 5,8-dimethoxylated isopimpinellin and bergapten had the smallest energy-minimized distance from the heme oxygen to the furan ring double bond. To evaluate this prediction, we expressed the CYP6AE89 protein in an Escherichia coli system, and used it to detect high catalytic activity against the two mono-methoxylated linear Furanocoumarins - bergapten and xanthotoxin - and weak activity against isopimpinellin. Thus, CYP6AE89, like CYP6AB3, is probably specialized for detoxifying only a subset of hostplant Furanocoumarins. A maximum-likelihood tree built with six representative lepidopterans with manually annotated cytochrome P450s shows that CYP6AE89 may have evolved much faster than the other CYP6AE proteins, possibly indicative of host selection pressure.
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cyp6b1 and cyp6b3 of the black swallowtail papilio polyxenes adaptive evolution through subfunctionalization
Molecular Biology and Evolution, 2006Co-Authors: Sanjeewa G Rupasinghe, May R Berenbaum, Mary A SchulerAbstract:Gene duplication provides essential material for functional divergence of proteins and hence allows organisms to adapt to changing environments. Following duplication events, redundant paralogs may undergo different evolutionary paths via processes known as nonfunctionalization, neofunctionalization, or subfunctionalization. Studies of adaptive evolution at the molecular level have progressed rapidly by computationally analyzing nucleotide substitution patterns but such studies are limited by the absence of information relating to alterations of function of the encoded enzymes. In this respect, evolution of the Papilio polyxenes cytochrome P450 monooxygenases (P450s) responsible for the adaptation of this insect to furanocoumarin-containing host plants provides an excellent model for elucidating the evolutionary fate of duplicated genes. Evidence from sequence and functional analysis in combination with molecular modeling indicates that the paralogous CYP6BI and CYP6B3 genes in P. polyxenes have probably evolved via subfunctionalization after the duplication event by which they arose. Both enzymes have been under independent purifying selection as evidenced by the low dN/dS ratio in both the coding region and substrate recognition sites. Both enzymes have maintained their ability to metabolize linear and angular Furanocoumarins albeit at different efficiencies. Comparisons of molecular models developed for the CYP6B3 and CYP6B1 proteins highlight differences in their binding modes that account for their different activities toward linear and angular Furanocoumarins. That P. polyxenes maintains these 2 furanocoumarin-metabolizing loci with somewhat different activities and expression patterns provides this species with the potential to acquire P450s with novel functions while maintaining those most critical to its exclusive feeding on its current range of host plants.
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characterization and evolution of furanocoumarin inducible cytochrome p450s in the parsnip webworm depressaria pastinacella
Insect Molecular Biology, 2004Co-Authors: W. Li, Arthur R. Zangerl, Mary A Schuler, May R BerenbaumAbstract:: Depressaria pastinacella, the parsnip webworm, a specialist on two genera in the Apiaceae, routinely consumes plant tissues high in furanocoumarin content and is capable of rapid cytochrome P450-mediated detoxification of these compounds. In this study, four cDNAs were cloned from the larval midgut of this insect: two full-length CYP6AB3 and CYP6AE1 cDNAs are closely related to members of the furanocoumarin-metabolizing CYP6B subfamily and two partial CYP9A6 and CYP9A7 cDNAs are related to members of the CYP9A subfamily that have also been linked to the detoxification of xenobiotics. At least one of these P450s (CYP6AB3) is inducible by dietary Furanocoumarins, indicating its potential involvement in furanocoumarin metabolism. A homology model of CYP6AB3 was constructed and compared to models of CYP6B1 from the specialist species, Papilio polyxenes, and CYP6B4 from the generalist species, P. glaucus. Structural superpositioning of these models has revealed very high spatial similarity of elements, including the B helix, B'-C loop, I helix and C-terminal domain, within the catalytic sites of these proteins. Most importantly, key amino acid residues that can potentially come into contact with furanocoumarin substrates display conservation in their spatial positioning and side chain polarities. Three of these residues, Val103, Leu113 and Phe118 (numbered according to CYP6AB3), are conserved in all three of these proteins, further implicating CYP6AB3 in furanocoumarin metabolism by parsnip webworms. Characterization of these P450 cDNAs will allow for functional analyses aimed at elucidating the molecular mechanisms underlying the coevolutionary interactions between this herbivore and its principal host plant.
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molecular analysis of cyp321a1 a novel cytochrome p450 involved in metabolism of plant allelochemicals Furanocoumarins and insecticides cypermethrin in helicoverpa zea
Gene, 2004Co-Authors: Masataka Sasabe, May R Berenbaum, Mary A SchulerAbstract:Abstract Cytochrome P450 monooxygenases play a significant role in the detoxification of hostplant allelochemicals and synthetic insecticides in Lepidoptera. In the corn earworm Helicoverpa zea , a noctuid of considerable economic importance, metabolisms of xanthotoxin, a toxic furanocoumarin, and α-cypermethrin, an insecticide, are mediated by at least one P450 with a catalytic site capable of accepting both substrates. To further the characterization of P450s in this species, we have cloned three full-length cDNAs encoding two CYP4M subfamily members and a novel CYP321A subfamily member. RNA analyses have demonstrated that the CYP321A1 gene is highly induced (51-fold) in larval midguts in response to xanthotoxin but not cypermethrin. Both CYP4M genes are expressed at negligible levels that are not increased by xanthotoxin or cypermethrin. Baculovirus-mediated expression of the full-length CYP321A1 cDNA has demonstrated that the CYP321A1 protein metabolizes xanthotoxin and angelicin, like the CYP6B1 protein in the furanocoumarin specialist Papilio polyxenes , and α-cypermethrin, like the CYP6B8 protein previously characterized in H. zea . In contrast, the CYP4M7 protein does not metabolize xanthotoxin at any detectable level. We conclude that at least two xanthotoxin-inducible P450s from highly divergent subfamilies (CYP6B and CYP321A) contribute to the resistance of H. zea larvae to toxic Furanocoumarins and insecticides. Genomic PCR analysis indicates that the CYP321A1 gene has evolved independently from the CYP6B genes known to be present in this insect.
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identification of variable amino acids in the srs1 region of cyp6b1 modulating furanocoumarin metabolism
Archives of Biochemistry and Biophysics, 2004Co-Authors: Liping Pan, May R Berenbaum, Jerome Baudry, Zhimou Wen, Mary A SchulerAbstract:The homology model of Papilio polyxenes CYP6B1 places Ile115, one of two variable amino acids, in the SRS1 of various CYP6B subfamily proteins in close proximity to the heme and Ala113, another variable amino acid, in a more distal position. We have constructed mutant CYP6B1 proteins altered at either of these positions and homology models of each based on multiple alignments with crystallized P450 proteins. The homology models suggest the existence of significant structural diversity in the hydrogen bond network surrounding the heme as a result of single point mutations in SRS1. Mutagenesis of Ile115 or Ala113 to other residues present in the insect CYP6B subfamily indicates that these amino acids control the spin state of the heme and, as a result, the catalytic activity of this monooxygenase. In particular, the I115L mutation significantly increases the spin state of the heme coordinately with 2- to 4-fold increases in its turnover of linear Furanocoumarins. Other A113V, A113L, A113Q, and A113E mutations display more variation in their effects but, in each case, strong correlations exist between furanocoumarin turnover and heme spin state. These data demonstrate that variable amino acids in SRS1 of the insect CYP6B subfamily exert dramatic effects on the range of Furanocoumarins metabolized, even when they occur in positions potentially distal from the substrate. These effects are possibly mediated through rearrangement of the local hydrogen bond network.
Arthur R. Zangerl - One of the best experts on this subject based on the ideXlab platform.
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characterization and evolution of furanocoumarin inducible cytochrome p450s in the parsnip webworm depressaria pastinacella
Insect Molecular Biology, 2004Co-Authors: W. Li, Arthur R. Zangerl, Mary A Schuler, May R BerenbaumAbstract:: Depressaria pastinacella, the parsnip webworm, a specialist on two genera in the Apiaceae, routinely consumes plant tissues high in furanocoumarin content and is capable of rapid cytochrome P450-mediated detoxification of these compounds. In this study, four cDNAs were cloned from the larval midgut of this insect: two full-length CYP6AB3 and CYP6AE1 cDNAs are closely related to members of the furanocoumarin-metabolizing CYP6B subfamily and two partial CYP9A6 and CYP9A7 cDNAs are related to members of the CYP9A subfamily that have also been linked to the detoxification of xenobiotics. At least one of these P450s (CYP6AB3) is inducible by dietary Furanocoumarins, indicating its potential involvement in furanocoumarin metabolism. A homology model of CYP6AB3 was constructed and compared to models of CYP6B1 from the specialist species, Papilio polyxenes, and CYP6B4 from the generalist species, P. glaucus. Structural superpositioning of these models has revealed very high spatial similarity of elements, including the B helix, B'-C loop, I helix and C-terminal domain, within the catalytic sites of these proteins. Most importantly, key amino acid residues that can potentially come into contact with furanocoumarin substrates display conservation in their spatial positioning and side chain polarities. Three of these residues, Val103, Leu113 and Phe118 (numbered according to CYP6AB3), are conserved in all three of these proteins, further implicating CYP6AB3 in furanocoumarin metabolism by parsnip webworms. Characterization of these P450 cDNAs will allow for functional analyses aimed at elucidating the molecular mechanisms underlying the coevolutionary interactions between this herbivore and its principal host plant.
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Host plant, host plant chemistry and the polyembryonic parasitoid Copidosoma sosares: indirect effects in a tritrophic interaction
Oikos, 2004Co-Authors: May R Berenbaum, Arthur R. Zangerl, Ian C.w. HardyAbstract:Host plant identity and host plant chemistry have often been shown to influence host finding and acceptance by natural enemies but comparatively less attention has been paid to the tritrophic effects of host plant and host plant chemistry on other natural enemy fitness correlates, such as survivorship, clutch size, body size, and sex ratio. Such studies are central to understanding both the selective impact of plants on natural enemies as well as the potential for reciprocal selective impact of natural enemies on plant traits. We examined the effects of host plant and host plant chemistry in a tritrophic system consisting of three apiaceous plants (Pastinaca sativa, Heracleum sphondylium and H. mantegazzianum), the parsnip webworm (Depressaria pastinacella) and the polyembryonic parasitic wasp Copidosoma sosares. All of these plants produce Furanocoumarins, known resistance factors for parsnip webworms. Furanocoumarin concentrations were correlated neither with the presence nor the number of webworms on a given plant. Concentrations of two Furanocoumarins were negatively associated with C. sosares fitness correlates: isopimpinellin with the likelihood that a given webworm would be parasitized and xanthotoxin with both within-brood survivorship (of all-male and mixed-sex broods) and clutch size. Brood sex ratio and body sizes of individual wasps were not correlated with furanocoumarin chemistry. Because additive genetic variation exists in P. sativa for furanocoumarin chemical traits, these are subject to selection by webworms through herbivory. Third trophic level selective impacts on furanocoumarin traits may include selection for reduced production of those chemicals that affect parasitoid survivorship yet do not influence host plant choice by the herbivore. That such might be the case is suggested by patterns of furanocoumarin production in populations of P. sativa with different histories of infestation; in the Netherlands, where parasitism rates of webworms by C. sosares are high, plants produce lower levels of all linear Furanocoumarins and proportionately less isopimpinellin than do midwestern U.S. populations of P. sativa, where natural enemies of the webworm are effectively absent.
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characterization of furanocoumarin metabolites in parsnip webworm depressaria pastinacella
Journal of Chemical Ecology, 2003Co-Authors: James K Nitao, Sandra M Duval, Mark A. Berhow, Arthur R. Zangerl, Steven F Vaughn, David Weisleder, May R BerenbaumAbstract:Although metabolites of Furanocoumarins have been characterized in a wide range of organisms, to date they have been identified in only a single insect species, Papilio polyxenes. Depressaria pastinacella, the parsnip webworm, like P. polyxenes a specialist on Apiaceae, routinely consumes plant tissues higher in furanocoumarin content than does P. polyxenes and is capable of faster cytochrome P-450-mediated detoxification of these compounds. In this study, we characterized metabolites of xanthotoxin, a linear furanocoumarin, and sphondin, an angular furanocoumarin, in midguts and frass of parsnip webworms. Two metabolites were isolated and identified from webworms fed artificial diet containing xanthotoxin. LC-ESI-MS analysis resulted in the determination of a MW of 266 for the compound in the frass and one of the compounds in the midgut; 1H NMR confirmed its structure as 6-(7-hydroxy-8-methoxycoumaryl)-hydroxyacetic acid (HCHA). The second compound from the midgut had a MW of 252 and was identified by 1H NMR and 13C NMR analysis as 6-(7-hydroxy-8-methoxycoumaryl)-hydroxyethanol) (HMCH). Whereas HCHA has been found in frass of Papilio polyxenes fed xanthotoxin, HMCH has not been reported previously in insects. Although the first step of metabolism of xanthotoxin in webworms as well as P. polyxenes is likely the formation of an epoxide on the furan ring, angular furanocoumarin metabolism in webworms appears to differ. The principal metabolite of sphondin was identified as demethylated sphondin (6-hydroxy-2H-furo[2,3-h]-1-benzopyran-2-one) by LC-ESI-MS and confirmed by 1H NMR and 13C NMR analyses. That webworms produce metabolites of xanthotoxin in common not only with other Lepidoptera (e.g., HCHA) but with other vertebrates (e.g., HMCH) suggests a remarkable conservatism in the metabolic capabilities of cytochrome P-450s and raises the possibility that insects may share other detoxification reactions with vertebrates with respect to toxins in foodplants.
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Effects of Furanocoumarins on feeding behavior of parsnip webworms Depressaria pastinacella
Journal of Chemical Ecology, 2002Co-Authors: J. A. Cianfrogna, Arthur R. Zangerl, May R BerenbaumAbstract:The parsnip webworm, Depressaria pastinacella, exhibits limited physiological resistance to furanocoumarin toxins in its principal host, the wild parsnip, Pastinaca sativa. These insects are typically found attacking individual plants low in Furanocoumarins, relative to others within populations. They also feed preferentially on parthenocarpic fruits, which are lower in furanocoumarin content than are normal fruits. However, in a previous study with artificial diets, they did not appear to discriminate between high and low concentrations of Furanocoumarins. In this study, the ability of webworms to distinguish between diets differing in furanocoumarin content was examined with an artificial diet containing wild parsnip and in green parsnip fruits with and without supplemental Furanocoumarins. Larvae showed no preference for high or low furanocoumarin diets containing equal amounts of freeze-dried parsnip fruit powder. When given a choice between otherwise similar wild parsnip fruits, webworms strongly preferred fruits that were not augmented with Furanocoumarins in one plant but showed no preference or only a weak preference for nonaugmented fruits in four other plants. In order to identify chemical constituents other than Furanocoumarins that might determine feeding preferences, we compared the chemical profile of parthenocarpic fruits (which are preferred) to that of normal fruit. Octyl butyrate, a known deterrent to webworms, is highly correlated with furanocoumarin content, occurs in all plants, and differs significantly among normal and parthenocarpic fruit, suggesting that webworms may be able to avoid Furanocoumarins by virtue of their behavioral response to octyl butyrate.
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expression of cyp6b1 and cyp6b3 cytochrome p450 monooxygenases and furanocoumarin metabolism in different tissues of papilio polyxenes lepidoptera papilionidae
Insect Biochemistry and Molecular Biology, 2001Co-Authors: Rebecca A Petersen, Arthur R. Zangerl, May R Berenbaum, Mary A SchulerAbstract:Abstract The CYP6B1 and CYP6B3 cytochrome P450 monooxygenases in the midgut of the black swallowtail participate in the metabolism of toxic Furanocoumarins present in its host plants. In this study, biochemical analyses indicate that the fat body metabolizes significant amounts of the linear Furanocoumarins bergapten and xanthotoxin after larvae feed on xanthotoxin. Northern analyses of the combined CYP6B1/3 transcript expression patterns indicate that transcripts in this P450 subfamily are induced in the midgut and fat body by xanthotoxin. Semi-quantitative RT–PCR analyses of individual CYP6B1/CYP6B3 mRNAs indicate that CYP6B1 transcripts are induced by xanthotoxin in all tissues examined and that CYP6B3 transcripts are induced in the fat body only. These results indicate that the fat body participates in the P450-mediated metabolism of excess Furanocoumarins unmetabolized by the midgut. Although transcripts of both genes were detected and CYP6B1 transcripts were induced by xanthotoxin in the integument, furanocoumarin metabolism was not detected. Comparison of these P450 promoters with the promoters of alcohol dehydrogenase genes expressed in the fat bodies of several Drosophila species suggest that the xanthotoxin inducibilities of these P450 genes in fat bodies are regulated by elements other than those modulating expression of Adh genes.
Frederic Bourgaud - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE The Distribution of Coumarins and Furanocoumarins in Citrus Species Closely Matches Citrus Phylogeny and Reflects the Organization of Biosynthetic Pathways
2016Co-Authors: Audray Dugr, Alain Hehn, Gilles Costantino, Patrick Ollitrault, Yann Froelicher, Re Olry, Frederic BourgaudAbstract:Citrus plants are able to produce defense compounds such as coumarins and furanocou-marins to cope with herbivorous insects and pathogens. In humans, these chemical com-pounds are strong photosensitizers and can interact with medications, leading to the “grapefruit juice effect”. Removing coumarins and Furanocoumarins from food and cosmet-ics imply additional costs and might alter product quality. Thus, the selection of Citrus culti-vars displaying low coumarin and furanocoumarin contents constitutes a valuable alternative. In this study, we performed ultra-performance liquid chromatography coupled with mass spectrometry analyses to determine the contents of these compounds within the peel and the pulp of 61 Citrus species representative of the genetic diversity all Citrus. Gen-erally, Citrus peel contains larger diversity and higher concentrations of coumarin/furano-coumarin than the pulp of the same fruits. According to the chemotypes found in the peel, Citrus species can be separated into 4 groups that correspond to the 4 ancestral taxa (pum-melos, mandarins, citrons and papedas) and extended with their respective secondary spe-cies descendants. Three of the 4 ancestral taxa (pummelos, citrons and papedas
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The distribution of coumarins and Furanocoumarins in Citrus species closely matches Citrus phylogeny and reflects the organization of biosynthetic pathways
2015Co-Authors: Audray Dugrand, Alain Hehn, Alexandre Olry, Yann Froelicher, Frederic BourgaudAbstract:In 2011, citrus crops represented one of the most important fruit productions in the world with more than 131 million tons. Citrus belong to the Rutaceae family able to produce coumarins which constitute a class of secondary metabolites commonly found in higher plants. In these plants, 7-hydroxycoumarin can undergo a subsequent two step enzymatic modification corresponding firstly to the prenylation at C6 and secondly to the closure of a furan ring, leading to Furanocoumarins. In parallel to ecological functions in plants, these molecules can be deleterious for humans. They are potential photosensitizers that can cause photophytodermatitis either after skin contact or ingestion followed by UV A exposure. This photosensitization property is a notable problem with Citrus essential oils, such as bergamot oil, since they are extensively used in perfumes. In this study, coumarin and furanocoumarin contents found in 61 citrus varieties are compared and the chemical diversity is discussed with respect to the genetic diversity. Based on hierarchical ascendant classification, coumarins and Furanocoumarins can be separated into 4 groups. Each of these 4 groups is logically organized with respect to the already described plant biosynthetic pathways and allows drawing hypotheses on the doubtful biosynthetic origin of compounds. With reference to Citrus diversity obtained from molecular markers, we highlight the genetic crossings which may have resulted in low coumarin and furanocoumarin content varieties. Citrus varieties identified in this study with low coumarin and furanocoumarin content will constitute invaluable genetic resources to breeding programs, promoting citrus species devoid of these toxic molecules.
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The distribution of coumarins and Furanocoumarins in Citrus species closely matches Citrus phylogeny and reflects the organization of biosynthetic pathways
PLoS ONE, 2015Co-Authors: Audray Dugrand-judek, Alain Hehn, Alexandre Olry, Gilles Costantino, Patrick Ollitrault, Yann Froelicher, Frederic BourgaudAbstract:Abstract Citrus plants are able to produce defense compounds such as coumarins and furanocou- marins to cope with herbivorous insects and pathogens. In humans, these chemical com- pounds are strong photosensitizers and can interact with medications, leading to the “grapefruit juice effect”. Removing coumarins and Furanocoumarins from food and cosmet- ics imply additional costs and might alter product quality. Thus, the selection of Citrus culti- vars displaying low coumarin and furanocoumarin contents constitutes a valuable alternative. In this study, we performed ultra-performance liquid chromatography coupled with mass spectrometry analyses to determine the contents of these compounds within the peel and the pulp of 61 Citrus species representative of the genetic diversity all Citrus. Gen- erally, Citrus peel contains larger diversity and higher concentrations of coumarin/furano- coumarin than the pulp of the same fruits. According to the chemotypes found in the peel, Citrus species can be separated into 4 groups that correspond to the 4 ancestral taxa (pum- melos, mandarins, citrons and papedas) and extended with their respective secondary spe- cies descendants. Three of the 4 ancestral taxa (pummelos, citrons and papedas) synthesize high amounts of these compounds, whereas mandarins appear practically devoid of them. Additionally, all ancestral taxa and their hybrids are logically organized according to the coumarin and furanocoumarin pathways described in the literature. This organization allows hypotheses to be drawn regarding the biosynthetic origin of compounds for which the biogenesis remains unresolved. Determining coumarin and furanocoumarin contents is also helpful for hypothesizing the origin of Citrus species for which the phylog- eny is presently not firmly established. Finally, this work also notes favorable hybridization schemes that will lead to low coumarin and furanocoumarin contents, and we propose toselect mandarins and Ichang papeda as Citrus varieties for use in creating species devoid of these toxic compounds in future breeding programs. Introduction PLOS
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A coumarin-specific prenyltransferase catalyzes the crucial biosynthetic reaction for furanocoumarin formation in parsley.
The Plant journal : for cell and molecular biology, 2014Co-Authors: Fazeelat Karamat, Alain Hehn, Frederic Bourgaud, Alexandre Olry, Ryosuke Munakata, Takao Koeduka, Akifumi Sugiyama, Cédric Paris, Kazufumi YazakiAbstract:Summary Furanocoumarins constitute a sub-family of coumarin compounds with important defense properties against pathogens and insects, as well as allelopathic functions in plants. Furanocoumarins are divided into two sub-groups according to the alignment of the furan ring with the lactone structure: linear psoralen and angular angelicin derivatives. Determination of furanocoumarin type is based on the prenylation position of the common precursor of all Furanocoumarins, umbelliferone, at C6 or C8, which gives rise to the psoralen or angelicin derivatives, respectively. Here, we identified a membrane-bound prenyltransferase PcPT from parsley (Petroselinum crispum), and characterized the properties of the gene product. PcPT expression in various parsley tissues is increased by UV irradiation, with a concomitant increase in furanocoumarin production. This enzyme has strict substrate specificity towards umbelliferone and dimethylallyl diphosphate, and a strong preference for the C6 position of the prenylated product (demethylsuberosin), leading to linear Furanocoumarins. The C8-prenylated derivative (osthenol) is also formed, but to a much lesser extent. The PcPT protein is targeted to the plastids in planta. Introduction of this PcPT into the coumarin-producing plant Ruta graveolens showed increased consumption of endogenous umbelliferone. Expression of PcPT and a 4–coumaroyl CoA 2'–hydroxylase gene in Nicotiana benthamiana, which does not produce Furanocoumarins, resulted in formation of demethylsuberosin, indicating that furanocoumarin production may be reconstructed by a metabolic engineering approach. The results demonstrate that a single prenyltransferase, such as PcPT, opens the pathway to linear Furanocoumarins in parsley, but may also catalyze the synthesis of osthenol, the first intermediate committed to the angular furanocoumarin pathway, in other plants.
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Coumarin and Furanocoumarin Quantitation in Citrus Peel via Ultraperformance Liquid Chromatography Coupled with Mass Spectrometry (UPLC-MS)
Journal of Agricultural and Food Chemistry, 2013Co-Authors: Audray Dugrand, Alain Hehn, Alexandre Olry, Yann Froelicher, Thibault Duval, Frederic BourgaudAbstract:Coumarins and Furanocoumarins are secondary metabolites commonly found in citrus plants. These molecules are allelochemical compounds in plants that have controversial effects on humans, such as phototoxicity and the commonly described interactions with drugs, referred to as the ``grapefruit juice effect''. Thus, it is important to develop a reliable method to identify and quantitate the coumarins and Furanocoumarins in citrus extracts. For this purpose, we herein describe an ultraperformance liquid chromatography coupled with mass spectrometry (UPLC-MS)-based method. We first developed a rapid UPLC method (20 min) to separate the isomers of each furanocoumarin. A subsequent single ion monitoring MS detection method was performed to distinguish between the molecules, which were possibly coeluting but had different molecular weights. The method was successfully used to separate and quantitate 6 coumarins and 21 Furanocoumarins in variable amounts within peel extracts (flayed and albedo) of 6 varieties of Citrus (sweet orange, lemon, grapefruit, bergamot, pummelo, and clementine). This method combines high selectivity and sensitivity in a rapid analysis and is useful for fingerprinting Citrus species via their coumarin and furanocoumarin contents.
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Comparative study of the production of coumarins and Furanocoumarins in three Ruteae species
Indian Journal of Natural Products and Resources, 2019Co-Authors: Saida Bergheul, Alain Hehn, Alexandre Olry, Mariana Limones-mendez, Jérémy Grosjean, Abdellah BerkaniAbstract:Within specialized metabolites, coumarins and Furanocoumarins represent a wide group of structurally diverse compounds and are specially produced in plants belonging to the Rutaceae family. Here we performed the furanocoumarin and coumarin-targeted chemical characterization of three Ruteae species collected from Algeria. Detection and quantification of 27 coumarins and Furanocoumarins extracted from stems and leaves was carried out by UHPLC-MS. We highlighted significant chemical differences between these plants. Ruta chalepensis L. is the highest producer with 24.83 mmol/g dry material in stems and 15.70 mmol/g dry materials in leaves while Haplophyllum tuberculatum ( Forsk.) is the lowest producer. We also showed a surprising chemical diversity between R . chalepensis L and R. angustifolia Pers . This chemical diversity might, therefore, be a helpful tool for phylogenetic identification of plants.
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A bacterial artificial chromosome (BAC) genomic approach reveals partial clustering of the furanocoumarin pathway genes in parsnip
Plant Journal, 2017Co-Authors: Sandro Roselli, Alexandre Olry, Guilhem Vialart, Sonia Vautrin, Olivier Coriton, David Ritchie, Gianni Galati, Nicolas Navrot, Celia Krieger, Helene BergesAbstract:Furanocoumarins are specialized metabolites that are involved in the defense of plants against phytophagous insects. The molecular and functional characterization of the genes involved in their biosynthetic pathway is only partially complete. Many recent reports have described gene clusters responsible for the biosynthesis of specialized metabolites in plants. To investigate possible co-localization of the genes involved in the furanocoumarin pathway, we sequenced parsnip BAC clones spanning two different gene loci. We found that two genes previously identified in this pathway, CYP71AJ3 and CYP71AJ4, were located on the same BAC, whereas a third gene, PsPT1, belonged to a different BAC clone. Chromosome mapping using fluorescence in situ hybridization (FISH) indicated that PsPT1 and the CYP71AJ3-CYP71AJ4 clusters are located on two different chromosomes. Sequencing the BAC clone harboring PsPT1 led to the identification of a gene encoding an Fe(II) alpha-ketoglutarate-dependent dioxygenase (PsDIOX) situated in the neighborhood of PsPT1 and confirmed the occurrence of a second gene cluster involved in the furanocoumarin pathway. This enzyme metabolizes p-coumaroyl CoA, leading exclusively to the synthesis of umbelliferone, an important intermediate compound in furanocoumarin synthesis. This work provides an insight into the genomic organization of genes from the furanocoumarin biosynthesis pathway organized in more than one gene cluster. It also confirms that the screening of a genomic library and the sequencing of BAC clones represent a valuable tool to identify genes involved in biosynthetic pathways dedicated to specialized metabolite synthesis.
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The distribution of coumarins and Furanocoumarins in Citrus species closely matches Citrus phylogeny and reflects the organization of biosynthetic pathways
2015Co-Authors: Audray Dugrand, Alain Hehn, Alexandre Olry, Yann Froelicher, Frederic BourgaudAbstract:In 2011, citrus crops represented one of the most important fruit productions in the world with more than 131 million tons. Citrus belong to the Rutaceae family able to produce coumarins which constitute a class of secondary metabolites commonly found in higher plants. In these plants, 7-hydroxycoumarin can undergo a subsequent two step enzymatic modification corresponding firstly to the prenylation at C6 and secondly to the closure of a furan ring, leading to Furanocoumarins. In parallel to ecological functions in plants, these molecules can be deleterious for humans. They are potential photosensitizers that can cause photophytodermatitis either after skin contact or ingestion followed by UV A exposure. This photosensitization property is a notable problem with Citrus essential oils, such as bergamot oil, since they are extensively used in perfumes. In this study, coumarin and furanocoumarin contents found in 61 citrus varieties are compared and the chemical diversity is discussed with respect to the genetic diversity. Based on hierarchical ascendant classification, coumarins and Furanocoumarins can be separated into 4 groups. Each of these 4 groups is logically organized with respect to the already described plant biosynthetic pathways and allows drawing hypotheses on the doubtful biosynthetic origin of compounds. With reference to Citrus diversity obtained from molecular markers, we highlight the genetic crossings which may have resulted in low coumarin and furanocoumarin content varieties. Citrus varieties identified in this study with low coumarin and furanocoumarin content will constitute invaluable genetic resources to breeding programs, promoting citrus species devoid of these toxic molecules.
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The distribution of coumarins and Furanocoumarins in Citrus species closely matches Citrus phylogeny and reflects the organization of biosynthetic pathways
PLoS ONE, 2015Co-Authors: Audray Dugrand-judek, Alain Hehn, Alexandre Olry, Gilles Costantino, Patrick Ollitrault, Yann Froelicher, Frederic BourgaudAbstract:Abstract Citrus plants are able to produce defense compounds such as coumarins and furanocou- marins to cope with herbivorous insects and pathogens. In humans, these chemical com- pounds are strong photosensitizers and can interact with medications, leading to the “grapefruit juice effect”. Removing coumarins and Furanocoumarins from food and cosmet- ics imply additional costs and might alter product quality. Thus, the selection of Citrus culti- vars displaying low coumarin and furanocoumarin contents constitutes a valuable alternative. In this study, we performed ultra-performance liquid chromatography coupled with mass spectrometry analyses to determine the contents of these compounds within the peel and the pulp of 61 Citrus species representative of the genetic diversity all Citrus. Gen- erally, Citrus peel contains larger diversity and higher concentrations of coumarin/furano- coumarin than the pulp of the same fruits. According to the chemotypes found in the peel, Citrus species can be separated into 4 groups that correspond to the 4 ancestral taxa (pum- melos, mandarins, citrons and papedas) and extended with their respective secondary spe- cies descendants. Three of the 4 ancestral taxa (pummelos, citrons and papedas) synthesize high amounts of these compounds, whereas mandarins appear practically devoid of them. Additionally, all ancestral taxa and their hybrids are logically organized according to the coumarin and furanocoumarin pathways described in the literature. This organization allows hypotheses to be drawn regarding the biosynthetic origin of compounds for which the biogenesis remains unresolved. Determining coumarin and furanocoumarin contents is also helpful for hypothesizing the origin of Citrus species for which the phylog- eny is presently not firmly established. Finally, this work also notes favorable hybridization schemes that will lead to low coumarin and furanocoumarin contents, and we propose toselect mandarins and Ichang papeda as Citrus varieties for use in creating species devoid of these toxic compounds in future breeding programs. Introduction PLOS
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A coumarin-specific prenyltransferase catalyzes the crucial biosynthetic reaction for furanocoumarin formation in parsley.
The Plant journal : for cell and molecular biology, 2014Co-Authors: Fazeelat Karamat, Alain Hehn, Frederic Bourgaud, Alexandre Olry, Ryosuke Munakata, Takao Koeduka, Akifumi Sugiyama, Cédric Paris, Kazufumi YazakiAbstract:Summary Furanocoumarins constitute a sub-family of coumarin compounds with important defense properties against pathogens and insects, as well as allelopathic functions in plants. Furanocoumarins are divided into two sub-groups according to the alignment of the furan ring with the lactone structure: linear psoralen and angular angelicin derivatives. Determination of furanocoumarin type is based on the prenylation position of the common precursor of all Furanocoumarins, umbelliferone, at C6 or C8, which gives rise to the psoralen or angelicin derivatives, respectively. Here, we identified a membrane-bound prenyltransferase PcPT from parsley (Petroselinum crispum), and characterized the properties of the gene product. PcPT expression in various parsley tissues is increased by UV irradiation, with a concomitant increase in furanocoumarin production. This enzyme has strict substrate specificity towards umbelliferone and dimethylallyl diphosphate, and a strong preference for the C6 position of the prenylated product (demethylsuberosin), leading to linear Furanocoumarins. The C8-prenylated derivative (osthenol) is also formed, but to a much lesser extent. The PcPT protein is targeted to the plastids in planta. Introduction of this PcPT into the coumarin-producing plant Ruta graveolens showed increased consumption of endogenous umbelliferone. Expression of PcPT and a 4–coumaroyl CoA 2'–hydroxylase gene in Nicotiana benthamiana, which does not produce Furanocoumarins, resulted in formation of demethylsuberosin, indicating that furanocoumarin production may be reconstructed by a metabolic engineering approach. The results demonstrate that a single prenyltransferase, such as PcPT, opens the pathway to linear Furanocoumarins in parsley, but may also catalyze the synthesis of osthenol, the first intermediate committed to the angular furanocoumarin pathway, in other plants.
