Distribution of lipoxygenase and hydroperoxide lyase in the leaves of various plant species

The enzyme activity responsible for volatile C₆-aldehyde formation was accompanied by lipoxygenase and hydroperoxide lyase in the green leaves of 28 plant species tested, but the level of each enzyme's activity varied. Lipoxygenase activity rather than hydroperoxide lyase activity appears to affect the overall C₆-aldehyde formation. There was a positive correlation (r = 0.712) between hydroperoxide lyase activity and the chlorophyll content of the green leaves; no correlation was found between lipoxygenase activity and chlorophyll content.     

Confounding of alternate respiration by lipoxygenase activity

The initial burst of respiratory activity (Q_o2) of imbibing soybean (Glycine max [L.] Merr. var. Wayne) seed tissue is cyanide-insensitive, and sensitive to salicylhydroxamate: presumptive evidence for the presence of alternate respiration. The initial O₂ consumption is also highly sensitive to propyl gallate. Soybean lipoxygenase exhibits similar characteristics of insensitivity to cyanide and sensitivity to salicylhydroxamate and to propyl gallate. The initial burst of respiration is enhanced by the addition of linoleic acid, a lipoxygenase substrate. These results indicate that the conventional tests for alternate respiration in plant tissues can be confounded by lipoxygenase; they also suggest that propyl gallate can be used to assess the possible participation of lipoxygenase in the O₂ uptake by plant tissues.     

Developmental change in c(6)-aldehyde formation by soybean leaves

Damage to plant leaves by wounding or freezing induces the production of large amounts of C₆-compounds. However, the control of formation of these compounds in leaves is not yet clear. In the current study, C₆-aldehyde formation by freeze-injured soybean leaves of different ages (based on the leaf positions on the plant) at stage R1 of plant development was investigated. The results demonstrate that C₆-aldehyde formation by the soybean (Glycine max L.) leaves changes as leaves develop. Younger leaves produce high levels of C₆-aldehydes, mainly composed of hexanal. Subsequently, as the leaves develop, the level of C₆-aldehyde formation decreases markedly, followed by an increase with a large shift from hexanal to hexenals. Lipoxygenase and lipolytic acyl hydrolase activity was reduced, and, in contrast, hydroperoxide lyase activity increased. There was little difference in lipoxygenase substrate specificity for linoleic acid and linolenic acid, but hydroperoxide lyase preferentially utilized 13-hydroperoxy-9,11,15-octadecatrienoic acid. In the in vivo lipoxygenase substrate pool, the linoleic acid level declined and the relative level of linolenic acid increased. The change in ratios of linolenic acid to linoleic acid showed a similar trend during soybean leaf development to that of hexenals to hexanal.     

Spontaneous Chemiluminescence of Soybean Embryonic Axes during Imbibition

Isolated soybean (Glycine max L. var Hood) embryonic axes have a spontaneous chemiluminescence (about 150 counts per minute per embryo) that increases showing two phases, upon water imbibition. The first photoemission burst was measured between 0 and 7 hours of imbibition with a maximum of about 350 counts per minute per embryo after 2 hours. The second photoemission phase, between 7 and 30 hours, increased from about 220 to 520 counts per minute per embryo. Both chemiluminescence phases were inhibited by infused butylated hydroxyanisole while only the second phase was inhibited by infused salicylhydroxamic acid. On the basis of the sensitivity of the lipoxygenase reaction to both inhibitors (about 90%), the first burst is tentatively assigned to oxy-radicals mobilized upon water uptake by the embryonic axes, and the second phase is tentatively identified as due to lipoxygenase activity. The in vivo lipoxygenase activity of the embryonic axes was estimated by both the fraction of total oxygen uptake that was inhibited by butylated hydroxyanisole and by the fraction of photoemission that was inhibited by butylated hydroxyanisole and by salicylhydroxamic acid. Both approaches indicated marked increases (5-fold and 12-fold, respectively) of lipoxygenase activity between 2 and 30 hours of imbibition. The measured chemiluminescence per O₂ uptake ratio (the experimental quantum yield) for the lipoxygenase reaction (3.3 × 10⁻¹⁴ counts per O₂ molecule) was used to estimate the O₂ uptake due to lipoxygenase activity from the photoemission of the embryonic axes after 30 hours of imbibition. The value (0.54 microliters per minute per axis) was close to the butylated hydroxyanisole-sensitive O₂ uptake (1.2 microliters O₂ per minute per axis) of the same embryonic axes. Chemiluminescence may afford a noninvasive assay for lipoxygenase activity in intact plant tissues.     

Investigation of the vascular actions of arachidonate lipoxygenase and cyclo-oxygenase products on the isolated perfused stomach of rat and rabbit

The vascular actions of several prostanoids and arachidonate lipoxygenase products were investigated on the gastric circulation of rat and rabbit perfused with Kreb's solution. Under resting conditions, prostacyclin and PGE₂ produced small decreases in perfusion pressure with prostacyclin being the more potent. During vasoconstriction induced by infusion of noradrenaline, vasopressin or angiotensin II, prostacyclin was 20–40 times as active as PGE₂ as a gastric vasodilator in rat or rabbit stomach. PGF_2α was a less potent vasoconstrictor than noradrenaline, while the epoxy-methano endoperoxide analogue produced a long-lasting vasoconstriction. The putative metabolite, 6-oxo-PGE₁ was less active than prostacyclin as a vasodilator, having comparable activity to PGE₁, whereas 6-oxo-PGF_1α had very little activity. The endoperoxide, PGH₂ reduced perfusion pressure, this effect being inhibited by concurrent infusion of 15-HPETE. The vasodilation induced by arachidonic acid was likewise reduced by 15-HPETE, and abolished by indomethacin infusion. The arachidonate lipoxygenase hydroperoxides were vasodilator in the gastric circulation, the rank order of potency being 12-HPETE > 11-HPETE > 5-HPETE > 15-HPETE in both rat and rabbit stomach. It is possible that such vasoactive lipoxygenase products, may play modulator roles in the gastric mucosa.     

Cyclic nucleotides modulate lipoxygenase activity and reproduction in oomycetes

Previous studies in our laboratory demonstrated that epoxy alcohols produced from C₂₀ fatty acids by lipoxygenase activity are associated with maintaining vegetative growth. We have also shown the specific down-regulation of lipoxygenase activity in reproductively competent oomycetes in response to cues which trigger reproduction. Other workers have suggested that cyclic nucleotides may also play a role in the switch between vegetative and reproductive growth of fungi. Reproductive activity was assayed in Achlya americana. Oogonium production was eliminated or reduced in the presence of cAMP, dibutyryl cAMP (0.5 mM), or the phosphodiesterase inhibitors caffeine (0.1, 1 mM) and theophylline (1 mM) and increased by 0.5 mM cGMP. Levels of cAMP were significantly higher in vegetative mycelium and were increased fourfold by exposure to 0.1 mM caffeine. These data suggest that an accumulation of cAMP inhibits reproductive activity while cGMP promotes it. Lipoxygenase activity was determined in the presence of cAMP, cAMP phosphodiesterase inhibitors, and cGMP to determine the interaction between cyclic nucleotides and lipoxygenase activity. Lipoxygenase activity in reproductive mycelium of A. americana or Saprolegnia ferax was reduced compared to vegetative mycelium. Lipoxygenase activity of cultures grown and starved in the presence of cAMP (0.5 mM) or caffeine (0.1 mM) showed significant increases over the comparably treated reproductive controls. Exposure to cGMP had no affect on lipoxygenase activity in A. americana. These data suggest that cAMP may maintain vegetative growth by maintaining relatively high lipoxygenase activity levels while the reproduction promoting effect of cGMP is not via lipoxygenase activity down-regulation. Adenylate cyclase activity was significantly higher in vegetative, compared to reproductive, mycelium of both A. americana and S. ferax. Elevated adenylate cyclase activity in vegetative mycelium supports the hypothesis that cAMP maintains vegetative growth by maintaining high lipoxygenase activity.     

Distinct lipoxygenase species appear in the hypocotyl/radicle of germinating soybean

Three lipoxygenase isozymes are synthesized in developing soybean (Glycine max [L.] Merr. cv Williams) embryos and are found in high levels in cotyledons of mature seeds (B Axelrod, TM Cheesbrough, S Zimmer [1981] Methods Enzymol 71: 441-451). Upon germination at least two new protein species appear which are localized mainly (on a protein basis) in the hypocotyl/radicle section. These lipoxygenase species appear also in seedlings of each of three lipoxygenase nulls (1×1, 1×2, and 1×3) deficient in one of the dormant seed lipoxygenases. The germination-associated species are distinguishable from dry seed lipoxygenase by their more acidic isoelectric points as revealed in isoelectric focusing gels. They are active from as early as 2 to at least 5 days after the start of imbibition. These germination-stimulated species qualify as lipoxygenase by their inhibition by the lipoxygenase inhibitors n-propyl gallate and salicyl hydroxamic acid and their lack of inhibition by KCN. Further, they are not active on the peroxidase substrate pair H₂O₂/3-amino-9-ethyl carbazole. They are recognized on Western blots by polyclonal antibodies to the seed lipoxygenase-1 isozyme and the major induced species has a molecular weight of approximately 100,000, similar to that of the cotyledon lipoxygenases. These lipoxygenases appear to be synthesized de novo upon germination since they comigrate with radioactive protein species from seeds germinated in [³⁵S]methionine.     

Inhibitory effect on soybean lipoxygenase and docking studies of some secondary metabolites,isolated from Origanum vulgare L. ssp. hirtum

In this study, five secondary metabolites (caffeic acid, rosmarinic acid, lithospermic acid B, 12-hydroxyjasmonic acid 12-O-β-glucoside and p-menth-3-ene-1,2-diol 1-O-β-glucopyranoside) isolated from the polar extracts of the plant Origanum vulgare L. ssp. hirtum, were tested in vitro for their ability to inhibit soybean lipoxygenase. Among the examined compounds, lithospermic acid B demonstrated the best inhibitory activity on soybean lipoxygenase with IC₅₀ = 0.1 mM. Docking studies have been undertaken as an attempt for better understanding the interactions of these compounds within the active site of soybean lipoxygenase. The predicted binding energy values correlated well with the observed biological data.     

Structural Features Required for Inhibition of Soybean Lipoxygenase-2 by Propyl Gallate : Evidence that Lipoxygenase Activity Is Distinct from the Alternative Pathway

The ability of 19 structural analogs of propyl gallate to inhibit purified soybean seed (Glycine max [L.] Merr. var. Ransom) lipoxygenase-2 (EC 1.13.11.12) was determined. The results indicate that the o-dihydroxy and not the ester function of propyl gallate is essential for inhibition of lipoxygenase. Catechol thus represents the minimum inhibitory structure. Among those compounds possessing an o-dihydroxy function, the K_i′ for inhibition of lipoxygenase is directly related to the lipophilicity of the inhibitor as measured by the octanol-water partition coefficient. The structural features of propyl gallate necessary for inhibition of lipoxygenase were found to differ from those required for inhibition of the plant mitochondrial alternative pathway. This further supports the concept that the alternative oxidase and lipoxygenase are functionally distinct species.     

Changes in lipoxygenase activity during seedling development of Lupinus albus

A lipoxygenase preparation was obtained from Lupinus albus seeds and was shown to differ from previously characterized lipoxygenase. This study describes changes in lipoxygenase activity during seedling development of Lupinus albus. The enzyme activity shows a decrease from 0–6 h postgermination (about 15%), is roughly constant or even rises slightly from 6–30 h and then shows a large increase between 30 and 48 h (about 50%). Enzymatically active proteins from 48 h-old seedlings were isolated and the increase of enzyme activity was mainly due to the presence of two components with maximum activity at pH 6 and pH 8.5, respectively. When arachidonic acid was used as substrate, the two enzymatic activities produce 15 HPETE. The increase in lipoxygenase activity during seedling development was inhibited by cycloheximide. Cordycepin appears to have no direct effect on lipoxygenase synthesis in vivo at the studied doses.     

Dicoumarol derivatives: Green synthesis and molecular modelling studies of their anti-LOX activity

Dicoumarol derivatives were synthesized in the InCl₃ catalyzed pseudo three-component reactions of 4-hydroxycoumarin with aromatic aldehydes in excellent yields. The reactions were performed in water under microwave irradiation. All synthesized compounds were characterized using NMR, IR, and UV–Vis spectroscopy, as well as with TD-DFT. Obtained dicoumarols were subjected to evaluation of their in vitro lipid peroxidation and soybean lipoxygenase inhibition activities. It was shown that five of ten examined compounds (3e, 3h, 3b, 3d, 3f) possess significant potential of antilipid peroxidation (84–97%), and that compounds 3b, 3e, 3h provided the highest soybean lipoxygenase (LOX-Ib) inhibition (IC₅₀ = 52.5 µM) and 3i somewhat lower activity (IC₅₀ = 55.5 µM). The bioactive conformations of the best LOX-Ib inhibitors were obtained by means of molecular docking and molecular dynamics. It was shown that, within the bioactive conformations interior to LOX-Ib active site, the most active compounds form the pyramidal structure made of two 4-hydroxycoumarin cores and a central phenyl substituent. This form serves as a spatial barrier which prevents LOX-Ib Fe²⁺/Fe³⁺ ion activity to generate the coordinative bond with the C13 hydroxyl group of the α-linoleate. It is worth pointing out that the most active compounds 3b, 3e, 3h and 3i can be candidates for further examination of their in vitro and in vivo anti-inflammatory activity and that molecular modeling study results provide possibility to screen bioactive conformations and elucidate the mechanism of dicoumarols anti-LOX activity.     

Molecular basis of seed lipoxygenase null traits in soybean line OX948

The poor stability and off-flavors of soybean oil and protein products can be reduced by eliminating lipoxygenases from soybean seed. Mature seeds of OX948, a lipoxygenase triple null mutant line, do not contain lipoxygenase proteins. The objective of this study was to determine the molecular basis of the seed lipoxygenase null traits in OX948. Comparisons of the sequences for lipoxygenase 1 (Lx1) and lipoxygenase 2 (Lx2) genes in the mutant (OX948) with those in a line with normal lipoxygenase levels (RG10) showed that the mutations in these genes affected a highly conserved group of six histidines necessary for enzymatic activity. The OX948 mutation in Lx1 is a 74?bp deletion in exon 8, which introduces a stop codon that prematurely terminates translation. A single T?CA substitution in Lx2 changes histidine H532 (one of the iron-binding ligands essential for L-2 activity) to glutamine. The mutation in the lipoxygenase 3 (Lx3) gene in OX948 is in the promoter region and represents two single base substitutions in a cis-acting AAATAC paired box. All three mutations would result in the loss of lipoxygenase activity in mature seed. The seed lipoxygenase gene mutation-based molecular markers could be used to accelerate and simplify breeding efforts for soybean cultivars with improved flavor.     

Differential modulation of the lipoxygenase cascade during typical and latent Pectobacterium atrosepticum infections

Background and AimsPlant diseases caused by Pectobacterium atrosepticum are often accompanied by extensive rot symptoms. In addition, these bacteria are able to interact with host plants without causing disease for long periods, even throughout several host plant generations. There is, to date, no information on the comparative physiology/biochemistry of symptomatic and asymptomatic plant–P. atrosepticum interactions. Typical (symptomatic) P. atrosepticum infections are associated with the induction of plant responses mediated by jasmonates, which are one of the products of the lipoxygenase cascade that gives origin to many other oxylipins with physiological activities. In this study, we compared the functioning of the lipoxygenase cascade following typical and latent (asymptomatic) infections to gain better insight into the physiological basis of the asymptomatic and antagonistic coexistence of plants and pectobacteria.MethodsTobacco plants were mock-inoculated (control) or infected with the wild type P. atrosepticum (typical infection) or its coronafacic acid-deficient mutant (latent infection). The expression levels of the target lipoxygenase cascade-related genes were assessed by Illumina RNA sequencing. Oxylipin profiles were analysed by GC-MS. With the aim of revising the incorrect annotation of one of the target genes, its open reading frame was cloned to obtain the recombinant protein, which was further purified and characterized using biochemical approaches.Key ResultsThe obtained data demonstrate that when compared to the typical infection, latent asymptomatic P. atrosepticum infection is associated with (and possibly maintained due to) decreased levels of 9-lipoxygenase branch products and jasmonic acid and increased level of cis-12-oxo-10,15-phytodienoic acid. The formation of 9-oxononanoic acid and epoxyalcohols in tobacco plants was based on the identification of the first tobacco hydroperoxide lyase (HPL) with additional epoxyalcohol synthase (EAS) activity.ConclusionsOur results contribute to the hypothesis of the oxylipin signature, indicating that different types of plant interactions with a particular pathogen are characterized by the different oxylipin profiles of the host plant. In addition, the tobacco LOC107825278 gene was demonstrated to encode an NtHPL (CYP74C43) enzyme yielding volatile aldehydes and aldoacids (HPL products) as well as oxiranyl carbinols (EAS products).     

Profiling of Volatile Compounds and Associated Gene Expression and Enzyme Activity during Fruit Development in Two Cucumber Cultivars

Changes in volatile content, as well as associated gene expression and enzyme activity in developing cucumber fruits were investigated in two Cucumis sativus L. lines (No. 26 and No. 14) that differ significantly in fruit flavor. Total volatile, six-carbon (C6) aldehyde, linolenic and linoleic acid content were higher during the early stages, whereas the nine-carbon (C9) aldehyde content was higher during the latter stages in both lines. Expression of C. sativus hydroperoxide lyase (CsHPL) mirrored 13-hydroperoxide lyase (13-HPL) enzyme activity in variety No. 26, whereas CsHPL expression was correlated with 9-hydroperoxide lyase (9-HPL) enzyme activity in cultivar No. 14. 13-HPL activity decreased significantly, while LOX (lipoxygenase) and 9-HPL activity increased along with fruit ripening in both lines, which accounted for the higher C6 and C9 aldehyde content at 0-6 day post anthesis (dpa) and 9-12 dpa, respectively. Volatile compounds from fruits at five developmental stages were analyzed by principal component analysis (PCA), and heatmaps of volatile content, gene expression and enzyme activity were constructed.     

Activation of Plasma Membrane NADH Oxidase Activity by Products of Phospholipase A

An auxin-stimulated NADH oxidase activity (NADH oxidase I) of plasma membrane vesicles, highly purified by aqueous two-phase partition from soybean (Glycine max Merr.) hypocotyls was activated by lysophospholipids and fatty acids, both products of phospholipase A action. The activation of NADH oxidase activity occurred slowly, suggesting a mechanism whereby the lipids acted to stabilize the enzyme in a more active configuration. In contrast to activation by lipids, the activation by auxin was rapid. The average K_m of the NADH oxidase after activation by lipids was four- to fivefold less than the K_m before activation. The V_max was unchanged by activation. The increases occurred in the presence of detergent and thus were not a result of exposure of latent active sites. Also, the activation did not result from activation of a peroxidase or lipoxygenase. Fatty acid esters, where growth promoting effects have been reported, also activated the auxin-stimulated oxidase. However, the auxin stimulation of NADH oxidase I did not appear to be obligatorily mediated by phospholipase A, nor did inhibitors of phospholipase A₂ block the stimulation of the oxidase by auxins.     

Study of the effect of volatile substances from cereal roots

We traced the liberation and biological effect of volatile substances released from the roots of cereals,i. e. barley, wheat, rye and oats, on seedlings of the same and other plant species. Experiments were carried out in a closed glass apparatus with a static or circulating atmosphere in which the CO₂ and O₂ were permanently absorbed and supplemented, respectively. In some experiments the air was bubbled through water or through solutions of boric acid, barium hydroxide and potassium permanganate. The roots of all four cereals tested released volatile substances with a biological activity which appeared to be non-specific with respect to plant species. The effect of volatile substances was partially decreased by bubbling through water, barium hydroxide and boric acid and was completely removed after passing through the solution of potassium permanganate. Volatile substances liberated from roots of barley inhibited elongation of roots and coleoptile, decreased SH-group content and caused excessive formation of root hairs as well as inhibition of both dry matter production and respiration of roots of rye seedlings. Ethylene was found in the atmosphere of experimental vessels.     

2-[(4″-Hydroxy-3′-methoxy)-phenoxy]-4-(4″-hydroxy-3″-methoxy-phenyl)-8-hydroxy-6-oxo-3-oxabicylo[3.3.0]-7-octene: unusual product of the soybean lipoxygenase-catalyzed oxygenation of curcumin

Racemic (1SR,2SR,4SR,5SR)-2-[(4′-hydroxy-3′-methoxy)-phenoxy]-4-(4″-hydroxy-3″-methoxy-phenyl)-8-hydroxy-6-oxo-3-oxabicyclo[3.3.0]-7-octene (2, C₂₁H₂₀O₈) was isolated as major product of soybean lipoxygenase action on curcumin (1, C₂₁H₂₀O₆). The structure of 2 was elucidated by HPLC-APCI-MS and tandem MS, ¹H, ¹³C, DEPT, H,H-COSY, H,C-HMQC, H,C-HMBC and phase sensitive 2D NOESY NMR techniques. For kinetic studies the rate of substrate degradation was followed spectrophotometrically at 430 nm, and the rate of oxygen consumption was measured polarographically. As evaluated by both methods, K_m for 1 was about four times higher than that obtained for linoleic acid (as the best substrate for soybean lipoxygenase); V_max was reduced five-fold. Lipoxygenase-mediated oxygenation of 1 was confirmed by the following criteria: (i) curcumin did not react with inactivated lipoxygenase; (ii) the enzymatic reaction was strongly inhibited by inhibitors such as BHA, deferoxamine and HgCl₂; (iii) oxygen consumption (measured polarographically) and curcumin degradation (measured photometrically) were shown to occur simultaneously at a ratio of 0.8 to 1, suggesting insertion of oxygen into 1 by lipoxygenase; (iv) molecular mass estimation by APCI-MS showed a shift of 32 in molecular mass from 1 (M_r 368) to 2 (M_r 400) being equivalent to an insertion of dioxygen. Curcumin meets none of the common features for lipoxygenase substrates and, therefore, may represent a new type of substrates for this enzyme.     

Inhibition of o(2) consumption resistant to cyanide and its development by N-propyl gallate and salicylhydroxamic Acid

Kinetics of inhibition of cyanide-insensitive O₂ uptake by n-propyl gallate (PG) and salicylhydroxamic acid (SHAM) were determined in fresh slices from ethylene-treated tubers of Solanum tuberosum `Norchip' and with mitochondria and lipoxygenase (EC 1.13.11.12) isolated from these tubers. PG and SHAM appeared to be inhibiting at identical sites in mitochondria but at disparate sites in slices. The apparent K_I for SHAM was similar in mitochondria and slices. However, the apparent K_I for PG in mitochondria was about 40-fold lower than the K_I for PG inhibition of lipoxygenase activity. The amount of lipoxygenase associated with mitochondria increased when tubers were treated with ethylene. PG, but not SHAM, inhibited aging-induced development of cyanide-insensitive respiration. The latter two phenomena are in accord with the hypothesis that lipid metabolism is required for the development of the alternative pathway.     

Interaction between Mitochondrial Cytochromes and Linoleic Acid Hydroperoxide: POSSIBLE CONFUSION WITH LIPOXYGENASE AND ALTERNATIVE PATHWAY

O₂ uptake by tissue extracts in the presence of linoleic acid is generally ascribed to lipoxygenase. Such an O₂ uptake can be observed not only with mitochondria of Solanum tuberosum L. and Arum maculatum L. and pure lipoxygenase but also with cytochrome c. However, the rate of oxidation is highly dependent on the procedure used to prepare the solutions of linoleic acid. Unless special care is taken to prevent contact between linoleic acid and O₂, it appears that linoleic acid hydroperoxide is readily formed. This derivative can be readily oxidized by mitochondria or cytochrome c. On the other hand, the use of a rapid and specific enzymic procedure to estimate the disappearance of linoleic acid demonstrates that linoleic acid itself is not consumed at any appreciable rate by mitochondria or cytochrome c, the true substrate being linoleic acid hydroperoxide. During the reaction, the heme nucleus of added cytochrome c or of mitochondrial cytochromes undergoes deep alterations. Therefore, caution should be exerted when equating an O₂ uptake observed in the presence of linoleic acid to a lipoxygenase activity. The same holds true for the similarity of reaction towards specific inhibitors between lipoxygenase and the cyanide-insensitive pathway oxidase.     

Ethane in pine needles preventing the feeding of the beetle, Monochamus alternatus

Since the remarkable repulsion activity for Monochamus alternatus of a gas from freshly ground needles of Pinus densiflora was observed by feeding tests, volatile components in the gas were submitted for further feeding tests. Among six components, five were known monoterpenic hydrocarbons, all of which showed a relatively low activity. The most abundant residual volatile in the gas was ethane, whose presence in gymnosperms has not been reported so far. Ethane showed a strong repulsion activity and was proved to be present also in the gases from other conifer needles of nine species but in less quantity than in the gas from the needles of P. densiflora. However, the order of repulsion due to various conifer needles was found to be roughly consistent with the order of ethane concentration in the gases from the respective needles. Saturated hydrocarbons with straight-chain C₅ to C₁₀ were also shown to be active for the beetle.