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Joerg Bohlmann – One of the best experts on this subject based on the ideXlab platform.

  • plasticity and evolution of 3 Carene synthase and sabinene synthase functions of a sitka spruce monoterpene synthase gene family associated with weevil resistance
    Journal of Biological Chemistry, 2014
    Co-Authors: Christopher R Roach, Dawn E Hall, Philipp Zerbe, Joerg Bohlmann
    Abstract:

    The monoterpene (+)-3Carene is associated with resistance of Sitka spruce against white pine weevil, a major North American forest insect pest of pine and spruce. High and low levels of (+)-3Carene in, respectively, resistant and susceptible Sitka spruce genotypes are due to variation of (+)-3Carene synthase gene copy number, transcript and protein expression levels, enzyme product profiles, and enzyme catalytic efficiency. A family of multiproduct (+)-3Carene synthase-like genes of Sitka spruce include the three (+)-3Carene synthases, PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and the (−)-sabinene synthase PsTPS-sab. Of these, PsTPS-3car2 is responsible for the relatively higher levels of (+)-3Carene in weevil-resistant trees. Here, we identified features of the PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and PsTPS-sab proteins that determine different product profiles. A series of domain swap and site-directed mutations, supported by structural comparisons, identified the amino acid in position 596 as critical for product profiles dominated by (+)-3Carene in PsTPS-3car1, PsTPS-3car2, and PsTPS-3car3, or (−)-sabinene in PsTPS-sab. A leucine in this position promotes formation of (+)-3Carene, whereas phenylalanine promotes (−)-sabinene. Homology modeling predicts that position 596 directs product profiles through differential stabilization of the reaction intermediate. Kinetic analysis revealed position 596 also plays a role in catalytic efficiency. Mutations of position 596 with different side chain properties resulted in a series of enzymes with different product profiles, further highlighting the inherent plasticity and potential for evolution of alternative product profiles of these monoterpene synthases of conifer defense against insects.

  • Plasticity and evolution of (+)-3Carene synthase and (-)-sabinene synthase functions of a sitka spruce monoterpene synthase gene family associated with weevil resistance.
    Journal of Biological Chemistry, 2014
    Co-Authors: Christopher R Roach, Dawn E Hall, Philipp Zerbe, Joerg Bohlmann
    Abstract:

    The monoterpene (+)-3Carene is associated with resistance of Sitka spruce against white pine weevil, a major North American forest insect pest of pine and spruce. High and low levels of (+)-3Carene in, respectively, resistant and susceptible Sitka spruce genotypes are due to variation of (+)-3Carene synthase gene copy number, transcript and protein expression levels, enzyme product profiles, and enzyme catalytic efficiency. A family of multiproduct (+)-3Carene synthase-like genes of Sitka spruce include the three (+)-3Carene synthases, PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and the (−)-sabinene synthase PsTPS-sab. Of these, PsTPS-3car2 is responsible for the relatively higher levels of (+)-3Carene in weevil-resistant trees. Here, we identified features of the PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and PsTPS-sab proteins that determine different product profiles. A series of domain swap and site-directed mutations, supported by structural comparisons, identified the amino acid in position 596 as critical for product profiles dominated by (+)-3Carene in PsTPS-3car1, PsTPS-3car2, and PsTPS-3car3, or (−)-sabinene in PsTPS-sab. A leucine in this position promotes formation of (+)-3Carene, whereas phenylalanine promotes (−)-sabinene. Homology modeling predicts that position 596 directs product profiles through differential stabilization of the reaction intermediate. Kinetic analysis revealed position 596 also plays a role in catalytic efficiency. Mutations of position 596 with different side chain properties resulted in a series of enzymes with different product profiles, further highlighting the inherent plasticity and potential for evolution of alternative product profiles of these monoterpene synthases of conifer defense against insects.

  • Biomarkers and gene copy number variation for terpenoid traits associated with insect resistance in Sitka spruce: An integrated genomic, proteomic, and biochemical analysis of (+)-3Carene biosynthesis
    BMC Proceedings, 2011
    Co-Authors: Joerg Bohlmann, Dawn E Hall, Jeanne A Robert, Christopher I Keeling, Dominik Domanski, Alfonso Lara Quesada, Sharon Jancsik, Michael A Kuzyk, Britta Hamberger, Christoph H Borchers
    Abstract:

    Conifers have evolved complex chemical defenses in the form of oleoresin terpenoids to resist attack from pathogens and herbivores. The large diversity of terpenoid metabolites is determined by the size and composition of the terpene synthase (TPS) gene family, and the single- and multi-product profiles of these enzymes. The monoterpene (+)-3Carene is associated with resistance of Sitka spruce (Picea sitchensis) to white pine weevil (Pissodes strobi). We used a combined genomic, proteomic and biochemical approach to analyze the (+)-3Carene phenotype in two contrasting Sitka spruce genotypes. Resistant trees produced significantly higher levels of (+)-3Carene than susceptible trees, in which only trace amounts were detected. Biosynthesis of (+)-3Carene is controlled, at the genome level, by a small family of closely related (82-95% amino acid sequence identity) (+)-3Carene synthase (PsTPS-3car) genes. Transcript profiling identified one PsTPS-3car gene (PsTPS-3car1) which is expressed in both genotypes, one gene (PsTPS-3car2) expressed only in resistant trees, and one gene (PsTPS-3car3) expressed only in susceptible trees. The PsTPS-3car2 gene was not detected in genomic DNA of susceptible trees. Target-specific selected reaction monitoring substantiated this pattern of differential expression of members of the PsTPS-3car family on the proteome level. Kinetic characterization of the recombinant PsTPS-3car enzymes identified differences in the activities of PsTPS-3car2 and PsTPS-3car3as a factor for the different (+)-3Carene profiles of resistant and susceptible trees. In conclusion, variation of the (+)-3Carene phenotype is controlled by PsTPS-3car gene copy number variation, variation of gene and protein expression, and variation of catalytic efficiencies.

Dawn E Hall – One of the best experts on this subject based on the ideXlab platform.

  • plasticity and evolution of 3 Carene synthase and sabinene synthase functions of a sitka spruce monoterpene synthase gene family associated with weevil resistance
    Journal of Biological Chemistry, 2014
    Co-Authors: Christopher R Roach, Dawn E Hall, Philipp Zerbe, Joerg Bohlmann
    Abstract:

    The monoterpene (+)-3Carene is associated with resistance of Sitka spruce against white pine weevil, a major North American forest insect pest of pine and spruce. High and low levels of (+)-3Carene in, respectively, resistant and susceptible Sitka spruce genotypes are due to variation of (+)-3Carene synthase gene copy number, transcript and protein expression levels, enzyme product profiles, and enzyme catalytic efficiency. A family of multiproduct (+)-3Carene synthase-like genes of Sitka spruce include the three (+)-3Carene synthases, PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and the (−)-sabinene synthase PsTPS-sab. Of these, PsTPS-3car2 is responsible for the relatively higher levels of (+)-3Carene in weevil-resistant trees. Here, we identified features of the PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and PsTPS-sab proteins that determine different product profiles. A series of domain swap and site-directed mutations, supported by structural comparisons, identified the amino acid in position 596 as critical for product profiles dominated by (+)-3Carene in PsTPS-3car1, PsTPS-3car2, and PsTPS-3car3, or (−)-sabinene in PsTPS-sab. A leucine in this position promotes formation of (+)-3Carene, whereas phenylalanine promotes (−)-sabinene. Homology modeling predicts that position 596 directs product profiles through differential stabilization of the reaction intermediate. Kinetic analysis revealed position 596 also plays a role in catalytic efficiency. Mutations of position 596 with different side chain properties resulted in a series of enzymes with different product profiles, further highlighting the inherent plasticity and potential for evolution of alternative product profiles of these monoterpene synthases of conifer defense against insects.

  • Plasticity and evolution of (+)-3Carene synthase and (-)-sabinene synthase functions of a sitka spruce monoterpene synthase gene family associated with weevil resistance.
    Journal of Biological Chemistry, 2014
    Co-Authors: Christopher R Roach, Dawn E Hall, Philipp Zerbe, Joerg Bohlmann
    Abstract:

    The monoterpene (+)-3Carene is associated with resistance of Sitka spruce against white pine weevil, a major North American forest insect pest of pine and spruce. High and low levels of (+)-3Carene in, respectively, resistant and susceptible Sitka spruce genotypes are due to variation of (+)-3Carene synthase gene copy number, transcript and protein expression levels, enzyme product profiles, and enzyme catalytic efficiency. A family of multiproduct (+)-3Carene synthase-like genes of Sitka spruce include the three (+)-3Carene synthases, PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and the (−)-sabinene synthase PsTPS-sab. Of these, PsTPS-3car2 is responsible for the relatively higher levels of (+)-3Carene in weevil-resistant trees. Here, we identified features of the PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and PsTPS-sab proteins that determine different product profiles. A series of domain swap and site-directed mutations, supported by structural comparisons, identified the amino acid in position 596 as critical for product profiles dominated by (+)-3Carene in PsTPS-3car1, PsTPS-3car2, and PsTPS-3car3, or (−)-sabinene in PsTPS-sab. A leucine in this position promotes formation of (+)-3Carene, whereas phenylalanine promotes (−)-sabinene. Homology modeling predicts that position 596 directs product profiles through differential stabilization of the reaction intermediate. Kinetic analysis revealed position 596 also plays a role in catalytic efficiency. Mutations of position 596 with different side chain properties resulted in a series of enzymes with different product profiles, further highlighting the inherent plasticity and potential for evolution of alternative product profiles of these monoterpene synthases of conifer defense against insects.

  • Biomarkers and gene copy number variation for terpenoid traits associated with insect resistance in Sitka spruce: An integrated genomic, proteomic, and biochemical analysis of (+)-3Carene biosynthesis
    BMC Proceedings, 2011
    Co-Authors: Joerg Bohlmann, Dawn E Hall, Jeanne A Robert, Christopher I Keeling, Dominik Domanski, Alfonso Lara Quesada, Sharon Jancsik, Michael A Kuzyk, Britta Hamberger, Christoph H Borchers
    Abstract:

    Conifers have evolved complex chemical defenses in the form of oleoresin terpenoids to resist attack from pathogens and herbivores. The large diversity of terpenoid metabolites is determined by the size and composition of the terpene synthase (TPS) gene family, and the single- and multi-product profiles of these enzymes. The monoterpene (+)-3Carene is associated with resistance of Sitka spruce (Picea sitchensis) to white pine weevil (Pissodes strobi). We used a combined genomic, proteomic and biochemical approach to analyze the (+)-3Carene phenotype in two contrasting Sitka spruce genotypes. Resistant trees produced significantly higher levels of (+)-3Carene than susceptible trees, in which only trace amounts were detected. Biosynthesis of (+)-3Carene is controlled, at the genome level, by a small family of closely related (82-95% amino acid sequence identity) (+)-3Carene synthase (PsTPS-3car) genes. Transcript profiling identified one PsTPS-3car gene (PsTPS-3car1) which is expressed in both genotypes, one gene (PsTPS-3car2) expressed only in resistant trees, and one gene (PsTPS-3car3) expressed only in susceptible trees. The PsTPS-3car2 gene was not detected in genomic DNA of susceptible trees. Target-specific selected reaction monitoring substantiated this pattern of differential expression of members of the PsTPS-3car family on the proteome level. Kinetic characterization of the recombinant PsTPS-3car enzymes identified differences in the activities of PsTPS-3car2 and PsTPS-3car3as a factor for the different (+)-3Carene profiles of resistant and susceptible trees. In conclusion, variation of the (+)-3Carene phenotype is controlled by PsTPS-3car gene copy number variation, variation of gene and protein expression, and variation of catalytic efficiencies.

Åke Ryrfeldt – One of the best experts on this subject based on the ideXlab platform.

  • Increased airway responsiveness of a common fragrance component, 3Carene, after skin sensitisation—a study in isolated guinea pig lungs
    Toxicology letters, 2003
    Co-Authors: Lena Låstbom, Anders Boman, Per Camner, S. Johnsson, Åke Ryrfeldt
    Abstract:

    Lungs from skin-sensitised and non-sensitised guinea pigs were exposed via the airways to 3Carene (1900 mg/m 3 ) and perfused with buffer containing either autologous plasma or lymphocytes. The experiments were performed in order to investigate the importance of blood components for the increased lung responsiveness seen in skin-sensitised animals. A reduction in lung function was noted in all lungs during 3Carene exposure. There was no difference in the 3Carene response between lungs from skin-sensitised animals versus lungs from non-sensitised animals when the perfusion buffer contained lymphocytes. However, when plasma diluted with buffer was used as perfusion medium, there was a significant enhancement in the response in lungs from sensitised versus lungs from non-sensitised animals. This implies that skin sensitisation increases lung responses to inhaled 3Carene and those components in plasma, and not the lymphocyte fraction, contributes to the observed increased lung responsiveness.

  • increased airway responsiveness of a common fragrance component 3 Carene after skin sensitisation a study in isolated guinea pig lungs
    Toxicology Letters, 2003
    Co-Authors: Lena Låstbom, Anders Boman, Per Camner, S. Johnsson, Åke Ryrfeldt
    Abstract:

    Lungs from skin-sensitised and non-sensitised guinea pigs were exposed via the airways to 3Carene (1900 mg/m 3 ) and perfused with buffer containing either autologous plasma or lymphocytes. The experiments were performed in order to investigate the importance of blood components for the increased lung responsiveness seen in skin-sensitised animals. A reduction in lung function was noted in all lungs during 3Carene exposure. There was no difference in the 3Carene response between lungs from skin-sensitised animals versus lungs from non-sensitised animals when the perfusion buffer contained lymphocytes. However, when plasma diluted with buffer was used as perfusion medium, there was a significant enhancement in the response in lungs from sensitised versus lungs from non-sensitised animals. This implies that skin sensitisation increases lung responses to inhaled 3Carene and those components in plasma, and not the lymphocyte fraction, contributes to the observed increased lung responsiveness.

  • Increased airway responsiveness after skin sensitisation to 3Carene, studied in isolated guinea pig lungs.
    Toxicology, 2000
    Co-Authors: Lena Låstbom, Anders Boman, Per Camner, Åke Ryrfeldt
    Abstract:

    Inhalation of 3Carene has been shown to induce bronchoconstriction in concentrations not far from the threshold limit value. In this study, one group of guinea-pigs were sensitised by dermal exposure to 3Carene according to the modified Cumulative Contact Enhancement Test protocol and another group of animals was used as controls. Lungs from the skin-sensitised and control guinea-pigs were perfused with diluted autologous blood (13 ml blood/87 ml buffer) and exposed to 3Carene at an air concentration of 3000 mg/m3. In both groups there was a reduction in compliance and conductance but this reduction was significantly (P