Diterpene Resin Acids Research Articles

Multicomponent Index for Evaluating Resistance by Sitka Spruce to the White Pine Weevil (Coleoptera: Curculionidae)

A prototype multicomponent resistance index was constructed for use in the selection of parent trees for a Sitka spruce, Picea sitchensis Bong (Carr.), breeding program and screening of progeny for resistance against the white pine weevil, Pissodes strobe (Peck). Data describing density of outer resin ducts, bark thickness, amount of volatile foliar terpenes, amount of cortical diterpene resin acids, feeding and oviposition deterrency, and repellency were collected from a clonal trial on Vancouver Island. These data were combined into a resistance index based on their hypothesized importance in weevil host selection. Clones differed in their complements of resistance traits, indicating that resistance is probably polygenic. There was a significant negative relationship between the resistance index and number of weevil attacks for the clones examined, suggesting that the resistance index could predict the resistance status of the trees. This relationship was improved if feeding deterrency and repellency were removed from the index. Our results suggest that a multicomponent resistance index is a promising tool for screening for resistance to P. strobi.

Isolation and characterization of isopimaric acid-degrading bacteria from a sequencing batch reactor.

We isolated two aerobic, gram-negative bacteria which grew on the diterpene resin acid isopimaric acid (IpA) as the sole carbon source and electron donor. The source of the isolates was a sequencing batch reactor treating a high-strength process stream from a paper mill. The isolates, IpA-1 and IpA-2, also grew on pimaric and dehydroabietic acids, and IpA-1 grew on abietic acid. Both strains used fatty acids, but neither strain used camphor, sitosterol, or betulin. Strain IpA-1 grew anaerobically with nitrate as an electron acceptor. Strains IpA-1 and IpA-2 had growth yields of 0.19 and 0.23 g of protein per g of IpA, respectively. During growth, both strains transformed IpA carbon to approximately equal amounts of biomass, carbon dioxide, and dissolved organic carbon. In both strains, growth on IpA induced an enzymatic system which caused cell suspensions to transform all four of the above resin acids. Cell suspensions of IpA-1 and IpA-2 removed IpA at rates of 0.56 and 0.13 mumol mg of protein-1 h-1, respectively. Cultures and cell suspensions of both strains failed to completely consume pimaric acid and yielded small amounts of an apparent metabolite from this acid. Cultures and cell suspensions of both strains yielded large amounts of three apparent metabolites from dehydroabietic acid. Analysis of 16S rDNA sequences indicated that the isolates are distinct members of the genus Pseudomonas sensu stricto.

Host plant influence on chemical defense in conifer sawflies (Hymenoptera: Diprionidae).

Host diet affects the defensive efficacy of Neodiprion sawflies. In laboratory assays with wood ants (Formica obscuripes), secretions from larvae reared on Pinus banksiana were the most repellent, while those from P. resinosa feeders were the least so. This was explained diterpene resin acid content, but not total monoterpene content. The terpene content of regurgitant generally reflected dietary concentrations. Compounds were sequestered nonselectively by larvae. Host-based differences in defense persisted at the behavioral level. P. banksiana feeders regurgitated greater volumes of fluid and were less likely to be disabled or killed by ants in one-on-one interactions than were larvae fed on P. resinosa. The defensive advantages of host diet conflicted with developmental requirements. N. sertifer reared on P. banksiana (the best diet for defense in all cases) had lower cocoon weights (a correlate of fecundity) than those reared on other diets, and had prolonged larval development compared to insects fed P. sylvestris. No such tradeoff was detected in N. lecontei. Larvae of both species strongly preferred P. banksiana over P. resinosa in feeding choice assays.

Induced oleoresin biosynthesis in grand fir as a defense against bark beetles.

Grand fir (Abies grandis) saplings and derived cell cultures are useful systems for studying the regulation of defensive oleoresinosis in conifers, a process involving both the constitutive accumulation of resin (pitch) in specialized secretory structures and the induced production of monoterpene olefins (turpentine) and diterpene resin acids (rosin) by nonspecialized cells at the site of injury. The pathways and enzymes involved in monoterpene and diterpene resin acid biosynthesis are described, as are the coinduction kinetics following stem injury as determined by resin analysis, enzyme activity measurements, and immunoblotting. The effects of seasonal development, light deprivation, and water stress on constitutive and wound-induced oleoresinosis are reported. Future efforts, including a PCR-based cloning strategy, to define signal transduction in the wound response and the resulting gene activation processes are delineated.

Fast atom bombardment (FAB) mass spectrometry of diterpene resin acid derivatives

The behaviour of methyl dehydroabietate and its ring C-substituted derivatives has been investigated by fast atom bombardment (FAB) mass spectrometry conditions. Similar FAB mass spectra were obtained in a 3-nitrobenzyl alcohol matrix using argon or xenon as the target gas, which has been attributed to the hydrophobic character of these compounds. Significant [M ‐ H] + , M +• and [M + H] + peaks and minor abundant [M ‐ 3H] + , [M ‐ 2H] +• and [M + 2H] +• ions have been observed to occur in the spectra. The intense M +• peaks generally observed have been rationalised in terms of the relatively aprotic and hydrophobic character of the matrix 3-nitrobenzyl alcohol and the hydrophobic nature of the compounds studied. The molecular ion represents the base peak in the molecular mass region of the amino abietates when 3-nitrobenzyl alcohol or triethyltetramine are used as matrices, presumably reflecting the stability of these radical cations. The relatively aprotic character of the matrix has been suggested to favour the ionization to M +• ions of the amino derivatives. The electrophilic nitro groups and/or a proton acceptor carbonyl group, have been found to play an important role in the formation of [M + H] + with respect to M +• ions. The enhanced abundance of [M + H] + with respect to M +• ions, when HCl was added to the matrix, as well as in a thioglycolic matrix, has been suggested to derive from a contribution of solution chemistry for the compounds bearing one to two unsubstituted amino groups. An important role played by the common hydrocarbon skeleton has been suggested to account for the significantly abundant [M ‐ H] + ions observed.

Antitumour lignans and cytotoxic resin acids from a New Zealand gymnosperm, Libocedrus plumosa

The antitumour activity of a foliage extract of a New Zealand gymnosperm, Libocedrus plumosa, against P388 leukaemia cells was due to β-peltatin 3. Deoxypodophyllotoxin 1 and β-peltatin A-methyl ether 2, P388-active lignans previously reported in L. bidwillii, were found at lower levels. High levels of trans-communic acid 4 and sandaracopimaric acid 5 were found in the L. plumosa extract. These diterpene resin acids showed cytotoxic activity against BSC-1 cells. A rapid "chemical screening" method is described, which further characterises bioactive extracts. Chemical screening and mass spectroscopy results for the two Libocedrus species and for Cupressus macrocarpa suggested that the P388 activity of the latter species was also due to 1 and 2. Extracts of all three of these species inhibited tubulin polymerisation.

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).

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.

Diterpene resin acids: Major active principles in tall oil against Variegated cutworm,Peridroma saucia (Lepidoptera: Noctuidae)

Tall oil, a by-product of the kraft process for pulping softwood, has been shown to have insecticidal properties. In the present study, the active principles in tall oil against the variegated cutworm,Peridroma saucia Hübner, were investigated. GC-MS analysis showed that abietic, dehydroabietic, and isopimaric acids were major resin acid components of crude tall oil and depitched tall oil. When crude tall oil samples of differing resin acid composition were incorporated into artificial diet at a concentration of 2.0% fresh weight, they suppressed larval growth by 45-60% compared to controls. This suppression was significantly (P≤0.05) correlated with the equivalent contents of abietic, dehydroabietic, isopimaric, and total resin acids. These results were also evident from a diet choice test, showing that the second-instar larvae obviously selected diets with low levels of resin acids when different diets were randomly arranged in a Petri dish. Bioassays with pure resin acids (abietic, dehydroabietic, and isopimaric acids) demonstrated that all individual chemicals have similar bioactivity against this insect. Comparison of the bioactivities of depitched tall oil and an equivalent mixture of pure resin acids in thePeridroma chronic growth bioassay indicated that pure resin acids and depitched tall oil share a common mode of action to this insect. This study confirms that resin acids are major active principles in tall oil against the variegated cutworm, but other chemicals likely also contribute to the bioactivity of tall oil.

Oleoresinosis in Grand Fir (Abies grandis) Saplings and Mature Trees (Modulation of this Wound Response by Light and Water Stresses).

The stem content of diterpene resin acids (rosin) increases dramatically following wounding of grand fir (Abies grandis) saplings, but the level of monoterpene olefins (turpentine) in the stem decreases following injury, in spite of a significant increase in monoterpene cyclase (synthase) activity. However, this observation was explained when rapid evaporative losses of the volatile monoterpenes from the wound site was demonstrated by trapping experiments, a finding consistent with a role of turpentine as a solvent for the mobilization and deposition of rosin to seal the injury. Mature forest trees responded to stem wounding by the enhancement of monoterpene cyclization capacity in a manner similar to 2-year-old grand fir saplings raised in the greenhouse. Light and water stresses greatly reduced the constitutive level of monoterpene cyclase activity and abolished the wound-induced response. The diminution in monoterpene biosynthetic capacity was correlated with a dramatic decrease in cyclase protein as demonstrated by immunoblotting. Relief of stress conditions resulted in the restoration of cyclase activity (both constitutive and wound induced) to control levels. The results of these experiments indicate that grand fir saplings are a suitable model for studies of the regulation of defensive oleoresinosis in conifers.

New Selective Synthesis of Oxidized Diterpene Resin Acid Derivatives

Abstract Cis 3 and trans methyl 7-oxo-podocarpa-8, 11, 13-trien-15-oate 4 are synthesized in high yields by photo-oxygenation of cis 1 and trans methyl podocarpa-8, 11, 13-trien-15-oate 2 obtained from dehydroabietic acid.

The influence of tree roots on spore germination of ectomycorrhizal fungi

Exudates from tree roots induce spore germination in ectomycorrhizal species of Suillus , whereas roots of herbs are mostly inactive in this respect. The active compound in pine roots was identified as abietic acid, a diterpene resin acid. The germination of the spores from several other ectomycorrhizal fungi was not affected by abietic acid. At high concentrations abietic acid increased the growth rate of Suillus granulatus and decreased that of S. luteus and Laccaria laccata .

Complete 13C and 1H NMR assignments for a series of aromatic C‐ring derivatives of dehydroabietic acid

AbstractComplete 13C and 1H NMR spectral assignments for a series of derivatives of the naturally occurring C‐ring aromatic diterpene resin acid dehydroabietic acid have been made. The use of a combination of 1D and 2D NMR techniques allowed mostly unambiguous assignments of all 13C resonances. 1H resonances were also assigned.

Effects of wounding on the terpene content of twigs of maritime pine (Pinus pinaster Ait.)

Summary. After wounding the cortical tissues of twigs of Pinus pinaster, we investigated changes in diterpene resin acid conterit and the ultrastructural modification of secretory structures. There was an increase in total resin acid content in the cortical and woody tissues located near the wound. Not all resin acids responded in the same way, but in wounded tissues the amounts of isopimaric acid and of the group of dehydroabietic, levopimarie and palustric acids significantly increased. The composition of cortical tissues becomes closely related to that of woody tissues. The resin acid enrichment of cortical tissues is correlated with the reactivation of the epithelial cells of the resin ducts and the de novo synthesis of resin acids demonstrated by labelling with J4CO2.

Specific effects of diterpene resin acids on spore germination of ectomycorrhizal basidiomycetes

Six diterpene resin acids out of eight tested possess the capacity to induce spore germination in all ten Swedish and American species ofSuillus tested. Species from other genera did not respond. A method for isolation of homokaryons (monosporous mycelia) ofSuillus species is described.

Biochemistry of Oleoresinosis

Elevated levels of monoterpenes and diterpene resin acids are produced in the stems of lodgepole pine (Pinus contorta var latifolia) saplings when wounded and inoculated with the blue-stain fungus Ceratocystis clavigera or when wounded and treated with a pectic fragment from tomato leaves (PIIF) or a fungal cell wall fragment (chitosan). This induced defensive response (hyperoleoresinosis) is the result of a transient rise in the ability to biosynthesize cyclic monoterpenes and diterpene resin acids as measured by the in vivo incorporation of label from [U-(14)C]sucrose relative to untreated controls, and is accompanied by a corresponding rise in the levels or activities of the relevant terpene cyclases as determined by in vitro assay using labeled acyclic precursors. The results indicate that juvenile P. contorta responds to infection and biotic elicitors much like the mature tree, and they suggest that the Pinaceae possess a mechanism for elicitor recognition and induced defense similar to that of other higher plants.

Abietic acid, and activator of basidiospore germination in ectomycorrhizal species of the genus Suillus (Boletaceae)

Germination of basidiospores from four ectomycorrhizaforming Suillus species ( S. granulatus, S. grevillei, S. luteus, and S. variegatus) was induced by exposing the spores to extracts or exudates from roots of Scotch pine ( Pinus sylvestris L.). An active compound was contained in the lipophilic phase obtained by extraction of pine roots with chloroform/methanol. It was isolated and identified as abietic acid, a diterpene resin acid. With S. granulatus, the bioassay species, abietic acid caused germination at concentrations down to 10 −7 M.

An evaluation of the differences in the composition of humic acids in soils under oak and pine forest by GC-MS after mild degradation

Significant differences were found in the products released after eight samples of humic acids (HA’s) isolated from soils under mediterranean pine and oak forests underwent mild degradation. A number of compounds, including typical degradation products of lignins, were found exclusively in the HA’s from the soils under pine forests. The aliphatic constituents were more firmly retained in the HA’s under oak, which presented mean values for aromatic acids lower than the humic samples from pine forests. Different types of diterpene resin acids were also found amongst the mild degradation products of HA’s from the studied areas.

Diterpene resin acids from the needle oleoresin of pinus strobus

The resin acid components of P. strobus needles were isolated, and the major constituents identified as labdenoic diterpenes. In addition to anticopalic and strobic acids previously reported in the needles, 3-oxoanticopalic, 3β-acetoxyanticopalic, 3β-hydroxyanticopalic acids and the 8α-hydroxy derivative of anticopalic acid were found along with two new compounds. The structures of the two new compounds, a cycloanticopalic acid and an abcoanticopalic acid, were determined by NMR. The composition of the diterpene resin acids was obtained for several samples of P. strobus needles from provenance tests.

A rapid method for diterpene resin acid analysis of pine needle oleoresin

A method for diterpene resin acid analysis has been developed for pine oleoresin samples at the submilligram level. The method utilizes the removal of acidic components by a micro-DEAE-Sephadex ion-exchange column and analysis of the recovered resin acids, as the methyl esters, by capillary gas–liquid chromatography.

The chemical feeding ecology ofNeodiprion dubiosus schedl,N. rugifrons midd., andN. lecontei (Fitch) on jack pine (Pinus banksiana lamb.)

Two monophagous sawflies,Neodiprion dubiosus andN. rugifrons, actively avoid feeding on the current season's needles of jack pine,Pinus banksiana, while the polyphagousN. lecontei feeds indiscriminately on foliage of all age classes. Bioassay studies with needle extracts indicate thatN. dubiosus andN. rugifrons larval feeding preference is governed by a group of tricyclic diterpene resin acids. Resin acids commonly occur in needles of all ages; however, levopimaric, palustric, dehydroabietic, and neoabietic acids were at higher concentrations in the new needles.N. dubiosus was significantly deterred from feeding on 1-year-old foliage when treated with the pure resin acids, palustric and levopimaric, at 0.5 and 1.0 mg/ml MeOH.N. rugifrons larvae were deterred from feeding by pure dehydroabietic, neoabietic, and palustric acids at 1.0 mg/ml MeOH.