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Rodney Croteau - One of the best experts on this subject based on the ideXlab platform.
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Novartis Foundation Symposium 223 - Insect-Plant Interactions and Induced Plant Defence - Diversity and variability of terpenoid defences in conifers: molecular genetics, biochemistry and evolution of the terpene synthase gene family in grand fir (Ab
Novartis Foundation symposium, 2007Co-Authors: Jörg Bohlmann, Rodney CroteauAbstract:This review focuses on the molecular genetics, biochemistry and evolution of terpenoid synthases relevant to terpenoid defences in conifers. In grand fir (Abies Grandis) biosynthesis of terpenoids of the three classes of monoterpenes, sesquiterpenes and diterpenes is inducible by stem wounding at the level of gene activation and increase of enzyme activity of the respective terpene synthases. The monoterpene, sesquiterpene and diterpene synthases utilize prenyl diphosphates of appropriate size as substrates to generate the large diversity of carbon skeletons characteristic of the terpenoid resin of conifers. A large and diverse gene family of grand fir terpene synthases has been cloned and cDNAs are actively expressed in Escherichia coli for enzyme characterization. The monophyletic group of grand fir monoterpene, sesquiterpene and diterpene synthases represents both constitutively expressed and inducible genes encoding single product and multiple product enzymes. Several events of gene duplication and functional specialization of new synthases occurred during the evolution of terpenoid biosynthesis in grand fir, and gave rise to the enormous diversity and variability of this ancient and successful plant defence against herbivores and pathogens. The review concludes with a perspective of the biotechnological applications of terpenoid synthases for the genetic engineering of agricultural crops and forest trees.
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Mutational analysis of a monoterpene synthase reaction: altered catalysis through directed mutagenesis of (-)-pinene synthase from Abies Grandis.
Archives of biochemistry and biophysics, 2005Co-Authors: David Hyatt, Rodney CroteauAbstract:Abstract Two monoterpene synthases, (−)-pinene synthase and (−)-camphene synthase, from grand fir ( Abies Grandis ) produce different product mixtures despite having highly homologous amino acid sequences and, presumably, very similar three-dimensional structures. The major product of (−)-camphene synthase, (−)-camphene, and the major products of (−)-pinene synthase, (−)-α-pinene, and (−)-β-pinene, arise through distinct mechanistic variations of the electrophilic reaction cascade that is common to terpenoid synthases. Structural modeling followed by directed mutagenesis in (−)-pinene synthase was used to replace selected amino acid residues with the corresponding residues from (−)-camphene synthase in an effort to identify the amino acids responsible for the catalytic differences. This approach produced an enzyme in which more than half of the product was channeled through an alternative pathway. It was also shown that several (−)-pinene synthase to (−)-camphene synthase amino acid substitutions were necessary before catalysis was significantly altered. The data support a model in which the collective action of many key amino acids, located both in and distant from the active site pocket, regulate the course of the electrophilic reaction cascade.
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Altering product outcome in Abies Grandis (−)-limonene synthase and (−)-limonene/(−)-α-pinene synthase by domain swapping and directed mutagenesis ☆
Archives of biochemistry and biophysics, 2004Co-Authors: Sadanobu Katoh, David Hyatt, Rodney CroteauAbstract:Abstract (−)-(4 S )-limonene synthase (LS) and (−)-(4 S )-limonene/(−)-(1 S , 5 S )-α-pinene synthase (LPS) from grand fir ( Abies Grandis ) exhibit nearly 91% sequence identity (93% similarity) at the amino acid level, yet produce very different mixtures of monoterpene olefins. To elucidate critical amino acids involved in determining monoterpene product distribution, a combination of domain swapping and reciprocal site-directed mutagenesis was carried out between these two enzymes. Exchange of the predicted helix D through F region in LS gave rise to an LPS-like product outcome, whereas reciprocal substitutions of four amino acids in LPS (two in the predicted helix D and two in the predicted helix F) altered the product distribution to that intermediate between LS and LPS, and resulted in a 5-fold increase in relative velocity. These results, in conjunction with modeling of the two enzymes, suggest that amino acids in the predicted D through F helix regions are critical for product determination.
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altering product outcome in Abies Grandis limonene synthase and limonene α pinene synthase by domain swapping and directed mutagenesis
Archives of Biochemistry and Biophysics, 2004Co-Authors: Sadanobu Katoh, David Hyatt, Rodney CroteauAbstract:Abstract (−)-(4 S )-limonene synthase (LS) and (−)-(4 S )-limonene/(−)-(1 S , 5 S )-α-pinene synthase (LPS) from grand fir ( Abies Grandis ) exhibit nearly 91% sequence identity (93% similarity) at the amino acid level, yet produce very different mixtures of monoterpene olefins. To elucidate critical amino acids involved in determining monoterpene product distribution, a combination of domain swapping and reciprocal site-directed mutagenesis was carried out between these two enzymes. Exchange of the predicted helix D through F region in LS gave rise to an LPS-like product outcome, whereas reciprocal substitutions of four amino acids in LPS (two in the predicted helix D and two in the predicted helix F) altered the product distribution to that intermediate between LS and LPS, and resulted in a 5-fold increase in relative velocity. These results, in conjunction with modeling of the two enzymes, suggest that amino acids in the predicted D through F helix regions are critical for product determination.
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geranyl diphosphate synthase from Abies Grandis cdna isolation functional expression and characterization
Archives of Biochemistry and Biophysics, 2002Co-Authors: Charles Burke, Rodney CroteauAbstract:Abstract Geranyl diphosphate synthase catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to generate geranyl diphosphate, the essential precursor of monoterpene biosynthesis. Using geranylgeranyl diphosphate synthase from Taxus canadensis as a hybridization probe, four full length cDNA clones, sharing high sequence identity to each other (>69%) and to the Taxus geranylgeranyl diphosphate synthase (>66%), were isolated from a grand fir ( Abies Grandis ) cDNA library. When expressed in Escherichia coli , three of the recombinant enzymes produced geranyl diphosphate and one produced geranylgeranyl diphosphate as the dominant product when supplied with isopentenyl diphosphate and dimethylallyl diphosphate as cosubstrates. One enzyme (AgGPPS2) was confirmed as a specific geranyl diphosphate synthase, in that it accepted only dimethylallyl diphosphate as the allylic cosubstrate and it produced exclusively geranyl diphosphate as product, with a k cat of 1.8 s −1 . Gel filtration experiments performed on the recombinant geranyl diphosphate synthases, in which the plastidial targeting sequences had been deleted, revealed that these enzymes are homodimers similar to other short-chain prenyltransferases but different from the heterotetrameric geranyl diphosphate synthase of mint.
Jörg Bohlmann - One of the best experts on this subject based on the ideXlab platform.
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Novartis Foundation Symposium 223 - Insect-Plant Interactions and Induced Plant Defence - Diversity and variability of terpenoid defences in conifers: molecular genetics, biochemistry and evolution of the terpene synthase gene family in grand fir (Ab
Novartis Foundation symposium, 2007Co-Authors: Jörg Bohlmann, Rodney CroteauAbstract:This review focuses on the molecular genetics, biochemistry and evolution of terpenoid synthases relevant to terpenoid defences in conifers. In grand fir (Abies Grandis) biosynthesis of terpenoids of the three classes of monoterpenes, sesquiterpenes and diterpenes is inducible by stem wounding at the level of gene activation and increase of enzyme activity of the respective terpene synthases. The monoterpene, sesquiterpene and diterpene synthases utilize prenyl diphosphates of appropriate size as substrates to generate the large diversity of carbon skeletons characteristic of the terpenoid resin of conifers. A large and diverse gene family of grand fir terpene synthases has been cloned and cDNAs are actively expressed in Escherichia coli for enzyme characterization. The monophyletic group of grand fir monoterpene, sesquiterpene and diterpene synthases represents both constitutively expressed and inducible genes encoding single product and multiple product enzymes. Several events of gene duplication and functional specialization of new synthases occurred during the evolution of terpenoid biosynthesis in grand fir, and gave rise to the enormous diversity and variability of this ancient and successful plant defence against herbivores and pathogens. The review concludes with a perspective of the biotechnological applications of terpenoid synthases for the genetic engineering of agricultural crops and forest trees.
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cDNA cloning, characterization, and functional expression of four new monoterpene synthase members of the Tpsd gene family from grand fir (Abies Grandis)
Archives of biochemistry and biophysics, 1999Co-Authors: Jörg Bohlmann, Sadanobu Katoh, Michael A. Phillips, Vasanthi Ramachandiran, Rodney CroteauAbstract:Abstract Grand fir (Abies Grandis) is a useful model system for studying the biochemistry, molecular genetics, and regulation of defensive oleoresin formation in conifers, a process involving both the constitutive accumulation of resin (pitch) in specialized secretory structures and the induced biosynthesis of monoterpenes and sesquiterpenes (turpentine) and diterpene resin acids (rosin) by nonspecialized cells at the site of injury. A similarity-based cloning strategy, employing primers designed to conserved regions of existing monoterpene synthases and anticipated to amplify a 1000-bp fragment, unexpectedly yielded a 300-bp fragment with sequence reminiscent of a terpenoid synthase. Utilization of this amplicon as a hybridization probe afforded four new, full-length cDNA species from a wounded fir stem cDNA library that appeared to encode four distinct monoterpene synthases. Expression in Escherichia coli, followed by enzyme assay with geranyl diphosphate (C10), farnesyl diphosphate (C15) and geranylgeranyl diphosphate (C20), and analysis of the terpene products by chiral phase gas chromatography and mass spectrometry confirmed that these sequences encoded four new monoterpene synthases, including (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, and an enzyme that produces both (−)-limonene and (−)-α-pinene. The deduced amino acid sequences indicated these enzymes to be 618 to 637 residues in length (71 to 73 kDa) and to be translated as preproteins bearing an amino-terminal plastid targeting sequence of 50–60 residues. cDNA truncation to delete the transit peptide allowed functional expression of the “pseudomature” forms of these enzymes, which exhibited no change in product outcome as a result of truncation. Sequence comparison revealed that these new monoterpene synthases from grand fir are members of the Tpsd gene subfamily and resemble sesquiterpene (C15) synthases and diterpene (C20) synthases from conifers more closely than mechanistically related monoterpene synthases from angiosperm species. The availability of a nearly complete set of constitutive and inducible monoterpene synthases from grand fir (now numbering seven) will allow molecular dissection of the resin-based defense response in this conifer species, and detailed study of structure–function relationships among this large and diverse family of catalysts, all of which exploit the same stereochemistry in the coupled isomerization–cyclization reaction.
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diversity and variability of terpenoid defences in conifers molecular genetics biochemistry and evolution of the terpene synthase gene family in grand fir Abies Grandis
Novartis Foundation symposium, 1999Co-Authors: Jörg Bohlmann, Rodney CroteauAbstract:This review focuses on the molecular genetics, biochemistry and evolution of terpenoid synthases relevant to terpenoid defences in conifers. In grand fir (Abies Grandis) biosynthesis of terpenoids of the three classes of monoterpenes, sesquiterpenes and diterpenes is inducible by stem wounding at the level of gene activation and increase of enzyme activity of the respective terpene synthases. The monoterpene, sesquiterpene and diterpene synthases utilize prenyl diphosphates of appropriate size as substrates to generate the large diversity of carbon skeletons characteristic of the terpenoid resin of conifers. A large and diverse gene family of grand fir terpene synthases has been cloned and cDNAs are actively expressed in Escherichia coli for enzyme characterization. The monophyletic group of grand fir monoterpene, sesquiterpene and diterpene synthases represents both constitutively expressed and inducible genes encoding single product and multiple product enzymes. Several events of gene duplication and functional specialization of new synthases occurred during the evolution of terpenoid biosynthesis in grand fir, and gave rise to the enormous diversity and variability of this ancient and successful plant defence against herbivores and pathogens. The review concludes with a perspective of the biotechnological applications of terpenoid synthases for the genetic engineering of agricultural crops and forest trees.
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Terpenoid-based defenses in conifers: cDNA cloning, characterization, and functional expression of wound-inducible (E)-alpha-bisabolene synthase from grand fir (Abies Grandis).
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Jörg Bohlmann, Reinhard Jetter, John Crock, Rodney CroteauAbstract:(E)-α-Bisabolene synthase is one of two wound-inducible sesquiterpene synthases of grand fir (Abies Grandis), and the olefin product of this cyclization reaction is considered to be the precursor in Abies species of todomatuic acid, juvabione, and related insect juvenile hormone mimics. A cDNA encoding (E)-α-bisabolene synthase was isolated from a wound-induced grand fir stem library by a PCR-based strategy and was functionally expressed in Escherichia coli and shown to produce (E)-α-bisabolene as the sole product from farnesyl diphosphate. The expressed synthase has a deduced size of 93.8 kDa and a pI of 5.03, exhibits other properties typical of sesquiterpene synthases, and resembles in sequence other terpenoid synthases with the exception of a large amino-terminal insertion corresponding to Pro81–Val296. Biosynthetically prepared (E)-α-[3H]bisabolene was converted to todomatuic acid in induced grand fir cells, and the time course of appearance of bisabolene synthase mRNA was shown by Northern hybridization to lag behind that of mRNAs responsible for production of induced oleoresin monoterpenes. These results suggest that induced (E)-α-bisabolene biosynthesis constitutes part of a defense response targeted to insect herbivores, and possibly fungal pathogens, that is distinct from induced oleoresin monoterpene production.
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Regulation of Oleoresinosis in Grand Fir (Abies Grandis) Differential Transcriptional Control of Monoterpene, Sesquiterpene, and Diterpene Synthase Genes in Response to Wounding
Plant physiology, 1998Co-Authors: Christopher L. Steele, Sadanobu Katoh, Jörg Bohlmann, Rodney CroteauAbstract:Grand fir (Abies Grandis Lindl.) has been developed as a model system for the study of wound-induced oleoresinosis in conifers as a response to insect attack. Oleoresin is a roughly equal mixture of turpentine (85% monoterpenes [C10] and 15% sesquiterpenes [C15]) and rosin (diterpene [C20] resin acids) that acts to seal wounds and is toxic to both invading insects and their pathogenic fungal symbionts. The dynamic regulation of wound-induced oleoresin formation was studied over 29 d at the enzyme level by in vitro assay of the three classes of synthases directly responsible for the formation of monoterpenes, sesquiterpenes, and diterpenes from the corresponding C10, C15, and C20 prenyl diphosphate precursors, and at the gene level by RNA-blot hybridization using terpene synthase class-directed DNA probes. In overall appearance, the shapes of the time-course curves for all classes of synthase activities are similar, suggesting coordinate formation of all of the terpenoid types. However, closer inspection indicates that the monoterpene synthases arise earlier, as shown by an abbreviated time course over 6 to 48 h. RNA-blot analyses indicated that the genes for all three classes of enzymes are transcriptionally activated in response to wounding, with the monoterpene synthases up-regulated first (transcripts detectable 2 h after wounding), in agreement with the results of cell-free assays of monoterpene synthase activity, followed by the coordinately regulated sesquiterpene synthases and diterpene synthases (transcription beginning on d 3–4). The differential timing in the production of oleoresin components of this defense response is consistent with the immediate formation of monoterpenes to act as insect toxins and their later generation at solvent levels for the mobilization of resin acids responsible for wound sealing.
Nicole L. Lang - One of the best experts on this subject based on the ideXlab platform.
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Tree invasion of a montane meadow complex: temporal trends, spatial patterns, and biotic interactions
2014Co-Authors: Charles B. Halpern, Joseph A. Antos, Janine M. Rice, Ryan D. Haugo, Nicole L. LangAbstract:Questions: Do spatial and temporal patterns of encroachment of Pinus contorta and Abies Grandis in a montane meadow suggest strong biotic controls on the invasion process? Location: Forest–meadow mosaic, 1350m a.s.l.
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Tree invasion of a montane meadow complex: temporal trends, spatial patterns, and biotic interactions
Journal of Vegetation Science, 2010Co-Authors: Charles B. Halpern, Joseph A. Antos, Janine M. Rice, Ryan D. Haugo, Nicole L. LangAbstract:Questions: Do spatial and temporal patterns of encroachment of Pinus contorta and Abies Grandis in a montane meadow suggest strong biotic controls on the invasion process? Location: Forest–meadow mosaic, 1350 m a.s.l., Cascade Range, Oregon, US. Methods: We combined spatial point pattern analysis, population age structures, and a time-series of stem maps to quantify spatial and temporal patterns of conifer invasion over a 200-yr period in three plots totaling 4 ha. Results: Trees established during two broad, but distinct periods (late 1800s, then at much greater density in the mid-1900s). Recent invasion was not correlated with climatic variation. Abies Grandis dominated both periods; P. contorta established at lower density, peaking before A. Grandis. Spatially, older (� 90 yr) P. contorta were randomly distributed, but older A. Grandis were strongly clumped (0.2-20 m). Younger (o90 yr) stems were positively associated at small distances (both within and between species), but were spatially displaced from older A. Grandis, suggesting a temporal shift from facilitation to competition. Establishment during the 1800s resulted in widely scattered P. contorta and clumps of A. Grandis that placed most areas of meadow close to seed sources permitting more rapid invasion during the mid-1900s. Rapid conversion to forest occurred via colonization of larger meadow openings – first by shade-intolerant P. contorta, then by shade-tolerant A. Grandis – and by direct infilling of smaller openings by A. Grandis. Conclusions: In combination, spatial and temporal patterns of establishment suggest an invasion process shaped by biotic interactions, with facilitation promoting expansion of trees into meadows and competition influencing subsequent forest development. Once invasion is initiated, tree species with different life histories and functional traits can interact synergistically to promote rapid conversion of meadow to forest under a broad range of climatic conditions.
Charles B. Halpern - One of the best experts on this subject based on the ideXlab platform.
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Tree invasion of a montane meadow complex: temporal trends, spatial patterns, and biotic interactions
2014Co-Authors: Charles B. Halpern, Joseph A. Antos, Janine M. Rice, Ryan D. Haugo, Nicole L. LangAbstract:Questions: Do spatial and temporal patterns of encroachment of Pinus contorta and Abies Grandis in a montane meadow suggest strong biotic controls on the invasion process? Location: Forest–meadow mosaic, 1350m a.s.l.
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Tree invasion of a montane meadow complex: temporal trends, spatial patterns, and biotic interactions
Journal of Vegetation Science, 2010Co-Authors: Charles B. Halpern, Joseph A. Antos, Janine M. Rice, Ryan D. Haugo, Nicole L. LangAbstract:Questions: Do spatial and temporal patterns of encroachment of Pinus contorta and Abies Grandis in a montane meadow suggest strong biotic controls on the invasion process? Location: Forest–meadow mosaic, 1350 m a.s.l., Cascade Range, Oregon, US. Methods: We combined spatial point pattern analysis, population age structures, and a time-series of stem maps to quantify spatial and temporal patterns of conifer invasion over a 200-yr period in three plots totaling 4 ha. Results: Trees established during two broad, but distinct periods (late 1800s, then at much greater density in the mid-1900s). Recent invasion was not correlated with climatic variation. Abies Grandis dominated both periods; P. contorta established at lower density, peaking before A. Grandis. Spatially, older (� 90 yr) P. contorta were randomly distributed, but older A. Grandis were strongly clumped (0.2-20 m). Younger (o90 yr) stems were positively associated at small distances (both within and between species), but were spatially displaced from older A. Grandis, suggesting a temporal shift from facilitation to competition. Establishment during the 1800s resulted in widely scattered P. contorta and clumps of A. Grandis that placed most areas of meadow close to seed sources permitting more rapid invasion during the mid-1900s. Rapid conversion to forest occurred via colonization of larger meadow openings – first by shade-intolerant P. contorta, then by shade-tolerant A. Grandis – and by direct infilling of smaller openings by A. Grandis. Conclusions: In combination, spatial and temporal patterns of establishment suggest an invasion process shaped by biotic interactions, with facilitation promoting expansion of trees into meadows and competition influencing subsequent forest development. Once invasion is initiated, tree species with different life histories and functional traits can interact synergistically to promote rapid conversion of meadow to forest under a broad range of climatic conditions.
Sadanobu Katoh - One of the best experts on this subject based on the ideXlab platform.
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Altering product outcome in Abies Grandis (−)-limonene synthase and (−)-limonene/(−)-α-pinene synthase by domain swapping and directed mutagenesis ☆
Archives of biochemistry and biophysics, 2004Co-Authors: Sadanobu Katoh, David Hyatt, Rodney CroteauAbstract:Abstract (−)-(4 S )-limonene synthase (LS) and (−)-(4 S )-limonene/(−)-(1 S , 5 S )-α-pinene synthase (LPS) from grand fir ( Abies Grandis ) exhibit nearly 91% sequence identity (93% similarity) at the amino acid level, yet produce very different mixtures of monoterpene olefins. To elucidate critical amino acids involved in determining monoterpene product distribution, a combination of domain swapping and reciprocal site-directed mutagenesis was carried out between these two enzymes. Exchange of the predicted helix D through F region in LS gave rise to an LPS-like product outcome, whereas reciprocal substitutions of four amino acids in LPS (two in the predicted helix D and two in the predicted helix F) altered the product distribution to that intermediate between LS and LPS, and resulted in a 5-fold increase in relative velocity. These results, in conjunction with modeling of the two enzymes, suggest that amino acids in the predicted D through F helix regions are critical for product determination.
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altering product outcome in Abies Grandis limonene synthase and limonene α pinene synthase by domain swapping and directed mutagenesis
Archives of Biochemistry and Biophysics, 2004Co-Authors: Sadanobu Katoh, David Hyatt, Rodney CroteauAbstract:Abstract (−)-(4 S )-limonene synthase (LS) and (−)-(4 S )-limonene/(−)-(1 S , 5 S )-α-pinene synthase (LPS) from grand fir ( Abies Grandis ) exhibit nearly 91% sequence identity (93% similarity) at the amino acid level, yet produce very different mixtures of monoterpene olefins. To elucidate critical amino acids involved in determining monoterpene product distribution, a combination of domain swapping and reciprocal site-directed mutagenesis was carried out between these two enzymes. Exchange of the predicted helix D through F region in LS gave rise to an LPS-like product outcome, whereas reciprocal substitutions of four amino acids in LPS (two in the predicted helix D and two in the predicted helix F) altered the product distribution to that intermediate between LS and LPS, and resulted in a 5-fold increase in relative velocity. These results, in conjunction with modeling of the two enzymes, suggest that amino acids in the predicted D through F helix regions are critical for product determination.
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cDNA cloning, characterization, and functional expression of four new monoterpene synthase members of the Tpsd gene family from grand fir (Abies Grandis)
Archives of biochemistry and biophysics, 1999Co-Authors: Jörg Bohlmann, Sadanobu Katoh, Michael A. Phillips, Vasanthi Ramachandiran, Rodney CroteauAbstract:Abstract Grand fir (Abies Grandis) is a useful model system for studying the biochemistry, molecular genetics, and regulation of defensive oleoresin formation in conifers, a process involving both the constitutive accumulation of resin (pitch) in specialized secretory structures and the induced biosynthesis of monoterpenes and sesquiterpenes (turpentine) and diterpene resin acids (rosin) by nonspecialized cells at the site of injury. A similarity-based cloning strategy, employing primers designed to conserved regions of existing monoterpene synthases and anticipated to amplify a 1000-bp fragment, unexpectedly yielded a 300-bp fragment with sequence reminiscent of a terpenoid synthase. Utilization of this amplicon as a hybridization probe afforded four new, full-length cDNA species from a wounded fir stem cDNA library that appeared to encode four distinct monoterpene synthases. Expression in Escherichia coli, followed by enzyme assay with geranyl diphosphate (C10), farnesyl diphosphate (C15) and geranylgeranyl diphosphate (C20), and analysis of the terpene products by chiral phase gas chromatography and mass spectrometry confirmed that these sequences encoded four new monoterpene synthases, including (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, and an enzyme that produces both (−)-limonene and (−)-α-pinene. The deduced amino acid sequences indicated these enzymes to be 618 to 637 residues in length (71 to 73 kDa) and to be translated as preproteins bearing an amino-terminal plastid targeting sequence of 50–60 residues. cDNA truncation to delete the transit peptide allowed functional expression of the “pseudomature” forms of these enzymes, which exhibited no change in product outcome as a result of truncation. Sequence comparison revealed that these new monoterpene synthases from grand fir are members of the Tpsd gene subfamily and resemble sesquiterpene (C15) synthases and diterpene (C20) synthases from conifers more closely than mechanistically related monoterpene synthases from angiosperm species. The availability of a nearly complete set of constitutive and inducible monoterpene synthases from grand fir (now numbering seven) will allow molecular dissection of the resin-based defense response in this conifer species, and detailed study of structure–function relationships among this large and diverse family of catalysts, all of which exploit the same stereochemistry in the coupled isomerization–cyclization reaction.
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Regulation of Oleoresinosis in Grand Fir (Abies Grandis) Differential Transcriptional Control of Monoterpene, Sesquiterpene, and Diterpene Synthase Genes in Response to Wounding
Plant physiology, 1998Co-Authors: Christopher L. Steele, Sadanobu Katoh, Jörg Bohlmann, Rodney CroteauAbstract:Grand fir (Abies Grandis Lindl.) has been developed as a model system for the study of wound-induced oleoresinosis in conifers as a response to insect attack. Oleoresin is a roughly equal mixture of turpentine (85% monoterpenes [C10] and 15% sesquiterpenes [C15]) and rosin (diterpene [C20] resin acids) that acts to seal wounds and is toxic to both invading insects and their pathogenic fungal symbionts. The dynamic regulation of wound-induced oleoresin formation was studied over 29 d at the enzyme level by in vitro assay of the three classes of synthases directly responsible for the formation of monoterpenes, sesquiterpenes, and diterpenes from the corresponding C10, C15, and C20 prenyl diphosphate precursors, and at the gene level by RNA-blot hybridization using terpene synthase class-directed DNA probes. In overall appearance, the shapes of the time-course curves for all classes of synthase activities are similar, suggesting coordinate formation of all of the terpenoid types. However, closer inspection indicates that the monoterpene synthases arise earlier, as shown by an abbreviated time course over 6 to 48 h. RNA-blot analyses indicated that the genes for all three classes of enzymes are transcriptionally activated in response to wounding, with the monoterpene synthases up-regulated first (transcripts detectable 2 h after wounding), in agreement with the results of cell-free assays of monoterpene synthase activity, followed by the coordinately regulated sesquiterpene synthases and diterpene synthases (transcription beginning on d 3–4). The differential timing in the production of oleoresin components of this defense response is consistent with the immediate formation of monoterpenes to act as insect toxins and their later generation at solvent levels for the mobilization of resin acids responsible for wound sealing.
