Publications

• Regulation of Oleoresinosis in Grand Fir (Abies grandis) (Coordinate Induction of Monoterpene and Diterpene Cyclases and Two Cytochrome P450-Dependent Diterpenoid Hydroxylases by Stem Wounding).

  Funk C., Lewinsohn E., Vogel B.S., Steele C.L., Croteau R.

  Oleoresin (pitch) is a defensive secretion composed of monoterpene olefins (turpentine) and diterpene resin acids (rosin) that is produced in grand fir (Abies grandis Lindl.) stems in response to wounding. Monoterpene and diterpene biosynthesis are coordinately induced in wounded stems as determin ... >> More

  Oleoresin (pitch) is a defensive secretion composed of monoterpene olefins (turpentine) and diterpene resin acids (rosin) that is produced in grand fir (Abies grandis Lindl.) stems in response to wounding. Monoterpene and diterpene biosynthesis are coordinately induced in wounded stems as determined by monitoring the activity of monoterpene and diterpene cyclases, as well as two cytochrome P450-dependent diterpenoid hydroxylases involved in the formation of ([mdash])-abietic acid, the principal resin acid of this species. The activity of these enzymes reaches maximum levels that are 5-to 100-fold higher than those of nowwounded control stems 10 d after wounding and this is followed by a synchronous decline. The increase in biosynthetic activity is consequently followed by the accumulation of a viscous mass of resin acids, with the loss of the volatile monoterpenes, at the site of injury. The observed coordinate induction of monoterpene olefin and abietic acid bio-synthesis and the results of oleoresin analysis are consistent with the role of the volatile monoterpenes as a solvent for the mobilization and deposition of resin acids at the wound site to seal the injury with a rosin barrier after the evaporation of the turpentine. The last step of resin acid biosynthesis is catalyzed by an operationally soluble aldehyde dehydrogenase that is not inducible by wounding but seemingly is expressed constitutively at a high level. In vivo [14C]acetate feeding and resin analysis indicate that this enzyme is not efficiently coupled to the earlier steps of the pathway. << Less

  Plant Physiol 106:999-1005(1994) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Diterpenoid resin acid biosynthesis in conifers: characterization of two cytochrome P450-dependent monooxygenases and an aldehyde dehydrogenase involved in abietic acid biosynthesis.

  Funk C., Croteau R.

  Abietic acid is a major component of the rosin fraction of oleoresin synthesized by grand fir (Abies grandis), lodgepole pine (Pinus contorta), and many other conifer species as a defensive secretion against insect and pathogen attack. The diterpenoid resin acid is derived from geranylgeranyl pyro ... >> More

  Abietic acid is a major component of the rosin fraction of oleoresin synthesized by grand fir (Abies grandis), lodgepole pine (Pinus contorta), and many other conifer species as a defensive secretion against insect and pathogen attack. The diterpenoid resin acid is derived from geranylgeranyl pyrophosphate via abietadiene, with subsequent oxidation of the C18-methyl of this olefin to abietadienol, abietadienal, and abietic acid. The pathway was confirmed by administering [1,2-14C]acetic acid to grand fir stems which incorporated the radiolabel into abietadiene, the corresponding alcohol and aldehyde, as well as abietic acid. Three different enzymatic activities, catalyzing the sequential oxidation of the olefin to abietic acid, were demonstrated in cell-free stem extracts of both grand fir and lodgepole pine. The first two oxidation steps were catalyzed by the microsomal fraction and required both oxygen and a reduced pyridine nucleotide (NADPH preferred). Both activities were strongly inhibited by CO (blue light reversible) and were differentially sensitive to several substituted N-heterocyclic inhibitors, suggesting that these two enzymes are distinct, microsomal cytochrome P450-dependent monooxygenases. A third enzymatic activity, catalyzing the oxidation of abietadienal to abietic acid, was located in the soluble protein fraction. This oxidation reaction employed NAD+ as cofactor, but did not require oxygen and was not inhibited by CO, indicating that this last step of abietic acid biosynthesis is catalyzed by an operationally soluble aldehyde dehydrogenase. << Less

  Arch Biochem Biophys 308:258-266(1994) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Loblolly pine abietadienol/abietadienal oxidase PtAO (CYP720B1) is a multifunctional, multisubstrate cytochrome P450 monooxygenase.

  Ro D.-K., Arimura G., Lau S.Y.W., Piers E., Bohlmann J.

  Cytochrome P450 monooxygenases (P450s) are important enzymes for generating some of the enormous structural diversity of plant terpenoid secondary metabolites. In conifers, P450s are involved in the formation of a suite of diterpene resin acids (DRAs). Despite their important role in constitutive ... >> More

  Cytochrome P450 monooxygenases (P450s) are important enzymes for generating some of the enormous structural diversity of plant terpenoid secondary metabolites. In conifers, P450s are involved in the formation of a suite of diterpene resin acids (DRAs). Despite their important role in constitutive and induced oleoresin defense, a P450 gene of DRA formation has not yet been identified. By using phylogenetic cluster analysis of P450-like ESTs from loblolly pine (Pinus taeda), functional cDNA screening in yeast (Saccharomyces cerevisiae), and in vitro enzyme characterization, we cloned and identified a multifunctional and multisubstrate cytochrome P450 enzyme, CYP720B1 [abietadienol/abietadienal oxidase (PtAO)]. PtAO catalyzes an array of consecutive oxidation steps with several different diterpenol and diterpenal intermediates in loblolly pine DRA biosynthesis. Recombinant PtAO oxidized the respective carbon 18 of abietadienol, abietadienal, levopimaradienol, isopimara-7,15-dienol, isopimara-7,15-dienal, dehydroabietadienol, and dehydroabietadienal with apparent Michaelis-Menten (K(m)) values of 0.5-5.3 muM. PtAO expressed in yeast also catalyzed in vivo oxidation of abietadiene to abietic acid, but with activity much lower than with abietadienol or abietadienal. Consistent with a role of DRAs in conifer defense, PtAO transcript levels increased upon simulated insect attack using methyl jasmonate treatment of loblolly pine. The multisubstrate, multifunctional P450 diterpene oxidase PtAO, in concert with expression of a family of single-product and multiproduct diterpene synthases, allows for formation of a diverse suite of DRA defense metabolites in long-lived conifers. << Less

  Proc. Natl. Acad. Sci. U.S.A. 102:8060-8065(2005) [PubMed] [EuropePMC]

  This publication is cited by 8 other entries.