Pine Extracts Research Articles

Diterpenoid Resin Acid Biosynthesis in Conifers: Characterization of Two Cytochrome P450-Dependent Monooxygenases and an Aldehyde Dehydrogenase Involved in Abietic Acid Biosynthesis

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.

Light-independent and light-dependent protochlorophyllide-reducing activities and two distinct NADPH-protochlorophyllide oxidoreductase polypeptides in mountain pine (Pinus mugo).

Lower plants and gymnosperms synthesize chlorophyll and develop photosynthetically competent chloroplasts even when grown in the dark. In cell-free extracts of pine (Pinus mugo, Turra, ssp. mugo) seedlings, light-independent and light-dependent protochlorophyllide-reducing activities are present. Two distinct NADPH-protochlorophyllide-oxidoreductase (POR) polypeptides can be detected immunologically with an antiserum raised against the POR of barley. The subcellular localization and amounts of the two POR polypeptides are differentially affected by light: one of them is predominantly present in prolamellar bodies of etiochloroplasts and its abundance rapidly declines once the pine seedlings are exposed to light; the other is found in thylakoid membranes and its amount does not change during illumination of dark-grown seedlings. Two types of cDNA sequences are identified that encode two distinct POR polypeptides in pine. The relevance of these POR polypeptides for the two chlorophyll biosynthetic pathways active in gymnosperms is discussed.

(1S,2R,4S,5S)-angelicoidenol-2-o-?-d-glucopyranoside?A moose deterrent compound in Scots pine (Pinus sylvestris L.)

Hydrophilic extracts of Scots pine (Pinus sylvestris L.) twigs have been investigated for palatability to moose in feeding experiments. The predominant repellent effect was observed from the 2-O-β-D-glucopyranoside of the monoterpene (1S,2R,4S,5S)-angelicoidenol. Of other isolated and tested substances, only taxifolin-3'-O-β-D-glucopyranoside had a deterring effect. One fraction-containing mainly aliphatic compounds-also had a strong effect, but the effect was lost after further fractionation. Tannin-containing fractions had no effect. Angelicoidenol glucoside has not previously been reported fromP. sylvestris. The results are discussed in view of phenols as defense compounds.

Glutamine synthetase in Scots pine seedlings and its control by blue light and light absorbed by phytochrome

The appearance of glutamine synthetase (GS. EC 6.3.1.2) in response to light and nitrogen (NO 3 (-) , NH 4 (+) ) was studied in the organs (roots, hypocotyl, cotyledonary whorl) of the Scots pine (Pinus sylvestris L.) seedling. Although GS activity was found to be mainly (> 80%) located in the whorl where it increased strongly in response to light, a significant GS synthesis was also detected in dark-grown seedlings. Anion-exchange chromatography was used to resolve two GS isoforms which appeared to be regulated differentially in the cotyledonary whorls. The isoform (presumably plastidic GS2) which eluted from the column at 90 mM KCl increased drastically in response to light. The other isoform (presumably cytosolic GS1), which eluted at 200 mM KCl, was not stimulated by light but tended to disappear during the experimental period (4 to 12 d after sowing). Immunoblotting of pine extract yielded a prominent band with a molecular weight of 43 kDa. The linear correlation between GS activity and immunodetectable GS protein could be extrapolated through zero, showing that any increase of GS2 activity is to be attributed to the de-novo synthesis of GS protein. Gelfiltration chromatography yielded a molecular mass for the GS holoenzyme of 340 kDa, a value which supports an octameric quarternary structure as previously suggested for angiosperms. While supplying seedlings with 10 mM NO 3 (-) stimulated GS synthesis in the whorl by 12%, 10 mM NH 4 (+) caused an incipient ammonium toxicity. Experiments using dischromatic light (simultaneous treatment with two light beams to vary the level of the physiologically active form of phytochrome, Pfr, in blue light) revealed that synthesis of GS2 was controlled by light in the same way as previously shown for ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1). Up to 10 d after sowing the strong light effect could be attributed to phytochrome action whereas between 10 and 12 d after sowing phytochrome control of GS-synthesis failed if no blue/ultraviolet-A light was provided. The data show that blue light is required to maintain responsiveness of GS2 synthesis to phytochrome. Both enzymes, GS2 as well as Fd-GOGAT, appear to be regulated coordinately to meet the demands of ammonium assimilation.

Acid‐Base Chemistry of Dissolved Organic Matter in Aqueous Leaf Extracts: Application to Organic Acids in Throughfall

AbstractElemental composition data were obtained for bulk precipitation and throughfall samples and for aqueous extracts of the leaves 6of three woody plant species common in the subalpine Sierra Nevada range, California: chinquapin (Chrysolepis sempervirens Hjelmqvist), western white pine (Pinus monticola Dougl.), and willow (Salix orestera Schneider). The acid‐base equilibria of the extracts were characterized by potentiometric titration and proton formation functions were computed. The latter then were modeled assuming four classes of quasiparticle acidic functional groups, yielding negative logarithms of conditional protonation constants in the range 4.8 to 5.0, 6.1 to 6.6, 7.4 to 7.7, and 9.1 to 9.4. The relative concentration of a given acidic functional group class varied markedly among the three woody species, but the conditional protonation constants were very similar. The model parameters, along with dissolved organic C concentration and pH values, were used to estimate net anion deficits in throughfall samples collected from the same sites as the leaf samples. On average, the calculated charge concentration of free organic anions in the western white pine extract matched the throughfall anion deficit, whereas the deficits in the chinquapin and willow throughfall samples were not accounted for by free anion concentrations. Metal complexation and in situ, species‐dependent leaf surface processes may account for these latter differences. In general, the anion deficit and, therefore, organic acids were an important component of rainfall and throughfall charge balance.

Pathogenicity and attraction to host extracts of Canadian pinewood nematodes: studies with Scots pine, western larch, and black spruce seedlings

Two isolates of the pinewood nematode, Bursaphelenchusxylophilus (Steiner and Buhrer), from Canada (an r form from Ontario and an m form from Quebec) and one from Japan (an r form) were inoculated into Scots pine, Pinussylvestris L., western larch, Larixoccidentalis Nutt., and black spruce, Piceamariana (Mill.) B.S.P. to determine the pathogenicity of this nematode and to observe changes in its numbers as well as the effect it had on seedling water and tannin content. Attractiveness of water, ethyl ether, and ethyl acetate stem extracts of these conifers to the pinewood nematode was also determined. The r form of the pinewood nematode was pathogenic on all three conifers: the Japanese isolate killed more Scots pine and the Ontario isolate, more black spruce. The Quebec isolate was as pathogenic as the other two isolates to western larch, weakly pathogenic to black spruce, and nonpathogenic to Scots pine. In general, there was a positive relationship between nematode population buildup and seedling mortality. No clear relationship was found between the water content of seedling stems and changes in nematode populations. Tannin content of stems increased following inoculation with pinewood nematode, but whereas correlation values between tannin content and nematode numbers were positive and significant, they were small. Although all three isolates were attracted to all host extracts, regardless of solvent used, ethyl acetate extracts were generally more attractive. The Japanese and Ontario isolates, which originated from pines, showed no particular affinity for Scots pine extracts; however, the Ontario isolate, which was especially attracted to black spruce extracts, was also most pathogenic to black spruce.

Determination of glutathione in Scots pine needles by high-performance liquid chromatography as its mono-bromobimane derivative

A method for determination of glutathione in its reduced (GSH) and oxidized (GSSG) forms in Scots pine extracts by reversed-phase high-performance liquid chromatography utilizing monobromobimane as the derivatization reagent was developed. The recovery and the precision for GSH in pine needles was high, ca. 99 and 3.6%, respectively. The determination of GSSG showed lower recovery ( ca. 80%) and poorer precision (13.3%). The identity of the putative GSH-bimane derivative was confirmed indirectly by gas chromatography-mass spectrometry. GSH comprised 77% of water-extractable thiols in pine needles, and the ratio GSSG/GSH was low (0.024).

Quantitative analysis of amino acids in conifer tissues by high-performance liquid chromatography and fluorescence detection of their 9-fluorenylmethyl chloroformate derivatives

Pre-column derivatization of amino acids in conifer extracts to form 9-fluorenylmethyl formate (FMOC) esters and the subsequent reversed-phase high-performance liquid chromatography (HPLC) analysis of these esters have been investigated. Qualitative analysis of putative FMOC-amino acid derivative HPLC peaks from a pine extract was performed by gas chromatography-mass spectrometry in order to confirm their identities. The precision, measured with ten replicate extracts, ranged from 2.2 to 15.6%. Theoretical responses from standards and extracts were compared with the actual response as an assay for systematic errors. The detector response was investigated as a function of the ratio amino acid/reagent and the range of linearity for each amino acid was established. The method is suitable for analysis of amino acids in crude extracts of small samples of Scots pine tissues, and for simultaneous determination of primary and secondary amino acids.

Inhibition of the Trichoderma viride cellulase complex by leaf litter extracts

The inhibition of the Trichoderma viride cellulase complex by extracts (hot water and 0.1 N NaOH) of deciduous and coniferous leaf litters in various stages of decomposition was investigated. Endocellulase and exocellulase activities were much more resistant to inhibition than β-glucosidase. White pine extracts were more inhibitory than deciduous ones and deciduous extracts had similar inhibitory potentials regardless of litter type. Except for β-glucosidase inhibition by water extracts, enzyme inhibition declined during decomposition. These results indicate that direct phenolic (humic) inhibition of cellulase components cannot account for species-specific patterns of cellulase activity in decomposing litter or for declining cellulase activity in the late stages of decomposition. Because β-glucosidase, the most readily inhibited component, might potentially limit cellulolysis, it was studied further, β-glucosidase inhibition by litter extracts was mitigated by the action of phenol oxidase and peroxidase. Complexing β-glucosidase with water-soluble litter fractions increased pH optima and Ea values, but did not affect apparent K m . Because of the mitigating effect of oxidative enzymes and presumed differences in extracellular localization of β-glucosidase relative to other cellulase components, β-glucosidase may not limit cellulolysis in situ.

An active component of pine leaves exhibiting anti-serotonergic action.

In examining the pharmacological effects of homeopathic medicines and other plant extracts on the contractile responses to serotonin in isolated rat aorta, the hexane : acetone (1 : 1) extract of pine leaves was found to possess an antiserotonergic effect. The extract was further fractionated by column chromatography to identify the active constituents. The results showed that polyprenol is the active component in pine leaves exhibiting anti-serotonergic action.

Allelopathic effects ofPinus densiflora on undergrowth of red pine forest

Correlation between the distributional frequency of undergrowth species of red pine forest and their germination and growth effected by pine extracts and leachates was found. It was made clear by germination and growth tests that pine toxic substances inhibit the germination and growth of low frequency species more than high frequency species in a red pine forest and that these substances are contained in descending concentration in fresh and fallen leaves, roots, pine forest soil, and pine rain. The concentration of pine toxic substances in extracts or leachates was affected by extracting or leaching within a given period of time, requiring a few hours for extracts or a few days for leachates. The amount of dry weight inhibition of the undergrowth treated by pine leachates was expressed as a growth inhibition index (GII) for the comparison of tolerance in various species. GII is a relative value (%) of the test groups against the control and it is an exponential function of the amount of pine toxic substances affecting the dry weight of the undergrowth. The substances were analyzed by paper and gas chromatography. Benzoic acid and 11 phenolic acids were identifed by gas chromatography. Benzoic acid was considered to be a key factor of allelopathy in the red pine forest.

Drywood termite feeding deterrents in sugar pine and antitermitic activity of related compounds

The feeding deterrent activity of sapwood extracts of sugar pine,Pinus lambertiana Dougl., and related compounds was determined against immatures of the western drywood termite,Incisitermes minor (Hagen). A bioassay was designed to quantify reductions in termite feeding caused by deterrent chemicals. Crude extracts and isolated fractions of sugar pine were deterrent and not preferred byI. minor at 0.5 mg/cm(2). Fatty acids occurring in sugar pine extracts had a broad range of deterrent activity. Long-chain saturated fatty acids were deterrent at 0.25 and 0.05 mg/cm(2). Unsaturated or intermediate length (C8-C14) acids, many not found in sugar pine wood, were less active than long-chain saturated acids. Related alpha-halogenated compounds were highly deterrent at 0.05 mg/cm(2) regardless of chain length or presence of a carboxylic acid moiety. Deposits of 2-iodooctadecanoic acid reduced termite feeding at 5 μg/cm(2), while 2-bromooctadecanoic acid had deterrent activity comparable to commercial wood preservatives. None of the halogenated compounds tested were termiticidal.

Croissance et fructification de Ceratocystis spp. avec attention particulière à C. piceae et C. fimbriata, en fonction du substrat

Ceratocystis coerulescens, C. fimbriata, C. ips, and C. minor were tested for production of sexual fruiting bodies, and C. penicillata and C. piceae for asexual fruiting bodies. Ceratocystis fimbriata produced perithecia easily on standard culture media, but there were marked differences between the two strains tested (503, 560). Strain 503 had a good production of fruiting bodies on malt agar (M) and a basal nutrient solution (N). Strain 560 fared better than 503 on Leonian agar (L), but did not fructify on M and N. Supplementing media with various wood extracts produced better results. M + maple sapwood extracts and L + poplar sapwood extracts gave the best results with strain 503, and L + pine sapwood extracts was the best with strain 560.Production of coremia was also influenced by the basal medium and the kind of extracts added as supplements. Fir and maple extracts stimulated the production of fruiting bodies, whereas pine and poplar extracts had no or very little stimulating effects. In every other species tested, the production of fruiting bodies was none or very irregular. [Journal translation]

GUSTATORY CUES IN THE ORIENTATION OF DENDROCTONUS PONDEROSAE (COLEOPTERA: SCOLYTIDAE) TO HOST TREES

AbstractFemale mountain pine beetles, Dendroctonus ponderosae Hopkins, were exposed to papers soaked in outer bark extracts of lodgepole pine, Pinus contorta Douglas var. latifolia Engelmann. Both benzene and methanol–water extracts elicited greater feeding activity than did controls. Non-polar host compounds exhibited greater incitant (initiation of feeding) properties, while polar compounds were more powerful stimulants (continuation of feeding). No differences were detected in feeding on extracts from resistant or susceptible trees. Repellency appears to be associated with beetle responses to active host metabolism.

Chromatography of acid phytohormones on columns of Sephadex LH‐20 and insoluble poly‐N‐vinylpyrrolidone, and application to the analysis of conifer extracts

The chromatographic behaviour of abscisic acid (ABA), indole‐3‐acetic acid (IAA), phenylacetic acid (PAA), and gibberellins A1, A4, A8, A9, A13 and A20 on columns of Sephadex LH‐20 and insoluble poly‐N‐vinylpyrrolidone (PVP) eluted with buffers of different pH values is described. PVP shows considerable batch differences that must be carefully checked. Chromatography of acidic ethyl acetate‐soluble fractions of Scots pine (Pinus sylvestris L.) extracts at pH 4.5 resulted in great losses of phytohormones, due to poor solubility of the extracts. If the extracts were applied to the column dissolved in buffer of pH 7.5, subsequent elution at pH 4.5 was possible with only small losses. The performance of the chromatographic column was strongly affected by the application volume. A combined PVP/Sephadex LH‐20 column eluted at pH 4.5 allows remarkable purification of pine and spruce (Picea abies (L.) Karst.) extracts, collection of IAA in a fraction that can be directly analyzed by e.g. the indolo‐α‐pyrone method, and collection of another fraction containing ABA, PAA and probably most of the known C19‐gibberellins; whereas the C20‐gibberellin A13 is eluted later (with IAA).

Identification of 3-indoleacetic acid in pinus sylvestris L. by gas chromatography-mas spectrometry, and quantitative analysis by ion-pair reversed-phase liquid chromatography wit spectrofluorimetric detection

The phytochormone 3-indoleacetic acid (IAA) was conclusively identified by gas chromatography-mas spetrometry of its methyl ester in purified extracts of Scots pine, Pinus sylvestris L. Preceding purification included reversed-phase ion-pair chromatography on a Nucleosil C18 column with tetrabutylammonium ion as counter ion. A number of indole acids were separated in this high-performance liquid chromatographic (HPLC) system. In particular, IAA was separated from the reported plant constituents 4-chloro-IAA, 5-hydroxy-IAA, 3-indoleacrylic acid, 3-indolelactic acid, 3-indolepropionic acid, 3-indolebutyric acid and tytophan. In connection with a spectrofluorine detector, the HPLC system was also used for the quantitative analysis of IAA in pine samples. Systematic errors caused a constant under-estimation of the IAA content by 12% and could easily be corrected for. The random error was 14%. Eight-week-old pine seedings contained 46 ng g−1 of IAA. The sensitivity of the method, applied to the analysis of pine extracts, was 50 pg of IAA. Evidence is presented that the method, when applied to the analysis of pine extracts, is specific of IAA.

Photosynthetic CO2 fixation in spinach chloroplasts inhibited by pine chloroplasts or extracts of pine chloroplasts

AbstractChloroplasts isolated from pine needles were found to be inactive with respect to CO2 fixation. Since it was suspected that pine needles may contain substances inhibitory to photosynthesis, studies were carried out using photosynthetically active isolated spinach chloroplasts and chloroplasts isolated from pine needles. When isolated pine chloroplasts were suspended in buffer and were added to isolated spinach chloroplasts they inhibited photosynthetic CO2 fixation. When the pine chloroplasts were separated from the medium by centrifugation, the separated pine chloroplasts severely inhibited CO2 fixation by isolated spinach chloroplasts, but the supernatant solution from the pine chloroplasts was not inhibitory. As little as 5% pine chloroplasts (based on chlorophyll content) produced 50% inhibition of CO2 fixation by the spinach chloroplasts. Studies of fixation of 14C‐labelled CO2 by spinach chloroplasts were carried out in which after 5 min photosynthesis the pine chloroplasts were added. It was found that the subsequent inhibition of spinach CO2 fixation was neither due to any effect on the rate of export of photosynthetic metabolites from the chloroplasts to the medium, nor to a direct effect on the RUBP carboxylase reaction. The principal effect was found to be an inhibition of the conversion of fructose‐1,6‐bisphosphate and sedoheptulose‐1,7‐bisphosphate to the respective monophosphates and inorganic phosphate. From this finding it was concluded that a principal effect of the inhibition by pine chloroplasts is probably an inhibition either directly or indirectly of the bisphosphatase enzymes in the spinach chloroplasts. Based on its distribution between organic and aqueous acidic or neutral solutions, the inhibitory factor of the pine chloroplasts must be lipophilic. Most of the factor could be transferred to an aqueous phase in a strongly alkaline solution. Following subsequent acidification of the aqueous phase the activity could be completely transferred back into the organic phase. This procedure allowed for separation of the inhibitory factor from most of the pigments and other lipophilic substances present in the pine chloroplasts and yielded a preparation which could be subsequently fractionated by thin layer chromatography. UV absorption was found in two fast moving spots and at the origin. The fastest running spot from the thin layer chromatography plate was found to be the one containing most of the inhibitory activity.

The chemical composition of aqueous extracts of coniferous needles

1. The group compositions of aqueous extracts of pine and spruce needles have been established. In their chemical compositions the extracts are similar to one another.

Using wood extracts to determine the feeding preferences of the western drywood termite,Incisitermes minor (Hagen)

Nymphs of the western drywood termite,Incisitermes minor (Hagen), were tested for their feeding preferences on wood and extracts of 11 tree species. The amount of wood consumed was inversely proportional to its specific gravity, but methanol extracts of least preferred woods were also least preferred when termites were confined to paper treated with extracts. In choice tests, only paper treated with extract of wood from which the colony developed and untreated and methanol-treated controls were significantly fed upon. Ponderosa pine extract applied to Douglas fir and sugar pine significantly decreased the amount of wood consumed. Resistance to termite feeding appeared to depend on the presence of repellent chemicals in the wood.

Properties of Phenylalanyl‐tRNA Synthetase and Phenylalanine Ammonia‐lyase of Pine Suspension Cultures

AbstractPhenylalanyl‐tRNA synthetase and phenylalanine ammonia‐lyase activities were demonstrated in partially purified extracts of pine (Pinus elliottii) suspension cultures. The optimum pH for the phenylalanyl‐tRNA synthetase reaction was 7.5 and the optimum ATP and Mg2+ concentrations were 1.0 and 15 mM respectively. Pine, calf liver and yeast tRNA were inadequate substitutes for pea tRNA in the synthetase reaction mixtures. The optimum pH for the phenylalanine ammonia‐lyase reaction was 9.0. The Km for phenylalanine was approximately 6.6 × 10−5M. The activity of both enzymes in the partially purified extracts was unstable on storage.