Signalling responses in the bark and foliage of canker-susceptible and -resistant cypress clones inoculated with Seiridium cardinale.

The necrotrophic fungus Seiridium cardinale is the main responsible for Cypress Canker Disease (CCD), a pandemic affecting many Cupressaceae worldwide. The present study aims to elucidate the signalling of the early responses in the bark and foliage of CCD-susceptible and -resistant C. sempervirens clones to S. cardinale inoculation (SI and RI, respectively). In the bark of SI, a peaking production of ethylene (Et) and jasmonic acid (JA) occurred at 3 and 4 days post inoculation (dpi), respectively, suggesting an attempted plant response to the pathogen. A response that, however, was ineffective, as confirmed by the severe accumulation of malondialdehyde by-products at 13 dpi (i.e., lipid peroxidation). Differently, Et emission peaked in RI bark at 3 and 13 dpi, whereas abscisic acid (ABA) accumulated at 1, 4 and 13 dpi, resulting in a lower MDA accumulation (and unchanged levels of antioxidant capacity). In the foliage of SI, Et was produced at 1 and 9 dpi, whereas JA and salicylic acid (SA) accumulated at 1 and 3 dpi. Conversely, an increase of ABA and SA occurred at 1 dpi in the RI foliage. This outcome indicates that some of the observed metabolic alterations, mainly occurring as local defence mechanisms, might be able to gradually shift to a systemic resistance, although an accumulation of MDA was observed in both SI and RI foliage (but with an increased antioxidant capacity reported only in the resistant clone). We believe that the results reported here will be useful for the selection of clones able to limit the spread and damage of CCD.

Essential oil composition and total phenolic content in Cupressus arizonica G. in response to microbial inoculation under water stress conditions.

Arizona Cypress is one of the drought-resistant, aromatic, and aesthetically pleasing trees having several pharmacological uses. Certain microorganisms contribute to the secondary metabolism and synthesis of bioactive compounds in aromatic and medicinal plants. This study aimed to determine the photosynthetic pigments, total phenolic content, antioxidant capacity, and essential oil composition of Arizona cypress under two irrigation regimes and microbial inoculations. We established a factorial experiment with three mycorrhizae inoculations (Rhizophagus irregularis, Funneliformis mosseae, and a mixture of R. irregularis and F. mosseae), a rhizobacterium inoculation (Pseudomonas fluorescens), and two irrigation regimes (well-watered and water stress). Under the water stress regime, seedlings inoculated with F. mosseae (0.46%) and non-inoculated control plants (0.29%) had the highest and lowest essential oil contents, respectively. GC-MS analysis revealed that limonen, a-pinene, terpinen-4-ol, and umbellulone were the most abundant compounds in the seedlings and treatments under study. The water stress regime had a significant and dominant effect on essential oil and antioxidant capacity, whereas seedling growth and photosynthetic pigments tended to decrease under stress conditions. However, co-inoculation of seedlings with mycorrhizae and the bacterium resulted in an increase in phenolic compounds and carotenoids. Under conditions of water stress and mycorrhizal symbiosis, the results of the current study may help increase the level of valuable compounds in Arizona cypress for further pharmaceutical applications.

Antimicrobial activity of biosynthesized silver nanoparticles, amoxicillin, and glass-ionomer cement againstStreptococcus mutansandStaphylococcus aureus.

Background. The development of dental caries is associated with various microorganisms and secondary caries formation is the main cause of restorations failure. The advice for restorative dental materials that have antimicrobial properties has stimulated the introduction of materials containing different antibacterial agents.Objectives. The present study has been designed to synthesize silver nanoparticles (AgNPs) and incorporate AgNPs and amoxicillin into glass ionomer cement (GIC) to synergize its effect on oral microbes. The effect of the added antimicrobial agents on compressive strength (CS) of GIC was also evaluated.Material and methods. Biosynthesis of AgNPs was done usingCupressus macrocarpaextract and AgNPs were characterized. A total of 120 disc-shaped specimens were prepared and classified into 4 main groups where Group A includes conventional GIC, Groups B and C include GIC with AgNPs or amoxicillin, respectively, while Group D included GIC with both AgNPs and amoxicillin. Each group was tested for the antimicrobial activity against bothStreptococcus mutans(S. mutans) andStaphylococcus aureus(S. aureus). The distribution of biofilm was examined via a scanning electron microscope. The CS of the tested material was measured using a Material Test System.Results. The UV-visible spectrum showed a peak of 429 nm. Transmission electron microscopy, x-ray diffraction pattern and Fourier transform infrared analysis confirmed the formation of AgNPs with spherical to oblong polydispersed particles of diameter in the range of 13.5-25.8 nm. The maximum inhibitory zone was recorded for group D against both tested bacteria with a mean of 29 mm at first 24 h period to 15 mm at three weeks and showed antimicrobial rate 92.2% and 92.56%, against both strains, respectively. Additionally, group D disintegrated the structure ofS. aureusbiofilm and even kill bacteria in the biofilms. The addition of AgNPs and amoxicillin caused an insignificant effect on CS of GIC.Conclusion.TheAgNPs showed a synergistic effect in combination with amoxicillin and GIC dental restorative material against studied microorganisms. The agents can be safely added with minimal effect on the mechanical properties of the original cement.

Cytotoxic and anti-inflammatory effects of lignans and diterpenes from Cupressus macrocarpa.

Cupressus macrocarpa is a windbreak tree and is reported to have various cytotoxic effects. A natural product study on the leaves of C. macrocarpa has yielded ten secondary metabolites, including three new diterpenoids (1-3), four known diterpenoids (4-7), and three known lignans (8-10). The structures of all isolated compounds were elucidated via the interpretation of spectroscopic methods, especially 2D NMR and mass analyses. In the cytotoxic assays, compounds 1-3 and 7-10 showed inhibition effect against HepG2, MDA-MB-231, and A549 cells with IC50 values ranging from 0.004 to 19.9 µg/mL. Moreover, the anti-inflammatory assays revealed that (-)-matairesinol (8) had significant inhibitory activities on superoxide anion generation (IC50 = 2.7 ± 0.3 µM) and elastase release (IC50 = 6.6 ± 0.7 µM).

Proton Gradient-Dependent Transport of p-Glucocoumaryl Alcohol in Differentiating Xylem of Woody Plants.

Lignin is a cell wall component of vascular plants crucial for survival in terrestrial environments. While p-hydroxyphenyl lignin is minor, it is considered to be localised in the outermost part of the cell wall providing strong adhesion between cells, which determines cell shape. Transport of the lignin precursor from the cytosol to the cell wall is critical to regulate temporal and spatial lignin deposition; however, little information on the transport step is available. Here, we report transport activity of p-glucocoumaryl alcohol, a precursor of p-hydroxyphenyl lignin, in a broad-leaved tree (hybrid poplar, Populus sieboldii × P. grandidentata) and a coniferous tree (Japanese cypress, Chamaecyparis obtusa). Membrane vesicles of both trees were prepared from differentiating xylem with vigorous lignification and used for transport assays. Several inhibition assays indicated that not ABC transporters but the proton gradient and V-ATPase are involved in p-glucocoumaryl alcohol transport depending on ATP. These results support the hypothesis that p-glucocoumaryl alcohol is loaded into the secretory vesicles and delivered to the cell wall by exocytosis. Furthermore, this transport mechanism was common in both poplar and Japanese cypress, strongly suggesting that p-glucocoumaryl alcohol transport in the differentiating xylem is conserved within woody plants.

Airborne Monoterpenes Emitted from a Cupressus lusitanica Cell Culture Induce a Signaling Cascade that Produces ß-Thujaplicin.

A cell culture of Cupressus lusitanica was used to investigate the reaction of a plant to certain airborne chemicals. Compared with laboratory and field methods using intact plants or tissues, a cell culture is advantageous because it is not affected by environmental factors, and the experiments are easier to reproduce. When exposed to an elicitor, our cell line produces 10 monoterpenes and ß-thujaplicin, which is a strong phytoalexin. These monoterpenes are emitted into the vapor phase and are expected to play a role in airborne signaling. In the present study, the cells were exposed to monoterpene vapors, and the volatiles present in the culture flasks were monitored. When the culture cells were exposed to low doses of sabinene, we detected γ-terpinene and p-cymene. After exposure to γ-terpinene, we found p-cymene and terpinolene, whereas p-cymene exposure resulted in terpinolene emission. By contrast, the other seven monoterpenes we investigated did not induce any emissions of other monoterpenes. These results strongly suggest that in C. lusitanica a signaling cascade exists that starts with the emission of sabinene and moves to γ-terpinene, p-cymene, and finally to terpinolene, which accelerates the production of the phytoalexin ß-thujaplicin.

Use of arbuscular mycorrhizal fungi to improve the drought tolerance of Cupressus atlantica G.

In this study, we investigated whether indigenous arbuscular mycorrhizal (AM) fungi could improve the tolerance of Cupressus atlantica against water deficit. We tested a gradient of watering regime spanning from 90% to 25% of soil retention capacity of water on mycorhized and non-mycorhized seedlings in pot cultures with sterilized and non-sterilized soils. Our result showed a positive impact of AM fungi on shoot height, stem diameter and biomass as well as on the growth rate. We also observed that inoculation with AM fungi significantly improved uptake of minerals by C. atlantica in both sterilized and non-sterilized soils independently of water regimes. We found that mycorhized plants maintained higher relative water content (RWC) and water potential compared with non-mycorhized plants that were subjected to drought-stress regimes (50% and 25% of soil retention capacity). The contents of proline and of soluble sugars showed that their concentrations decreased in non-mycorhized plants subjected to DS. Superoxide dismutase (SOD) and catalase (CAT) activities also decreased in non-mycorhized plants submitted to DS compared to mycorhized plants. The same pattern was observed by measuring peroxidase (POD) enzyme activity. The results demonstrated that AM fungal inoculation promoted the growth and tolerance of C. atlantica against DS in pot cultures. Therefore, mycorrhizal inoculation could be a potential solution for the conservation and reestablishment of C. atlantica in its natural ecosystem.

Chromium Biosorption from Cr(VI) Aqueous Solutions by Cupressus lusitanica Bark: Kinetics, Equilibrium and Thermodynamic Studies.

The present study investigated the kinetics, equilibrium and thermodynamics of chromium (Cr) ion biosorption from Cr(VI) aqueous solutions by Cupressus lusitanica bark (CLB). CLB total Cr biosorption capacity strongly depended on operating variables such as initial Cr(VI) concentration and contact time: as these variables rose, total Cr biosorption capacity increased significantly. Total Cr biosorption rate also increased with rising solution temperature. The pseudo-second-order model described the total Cr biosorption kinetic data best. Langmuir´s model fitted the experimental equilibrium biosorption data of total Cr best and predicted a maximum total Cr biosorption capacity of 305.4 mg g(-1). Total Cr biosorption by CLB is an endothermic and non-spontaneous process as indicated by the thermodynamic parameters. Results from the present kinetic, equilibrium and thermodynamic studies suggest that CLB biosorbs Cr ions from Cr(VI) aqueous solutions predominantly by a chemical sorption phenomenon. Low cost, availability, renewable nature, and effective total Cr biosorption make CLB a highly attractive and efficient method to remediate Cr(VI)-contaminated water and wastewater.

Strong Induction of Minor Terpenes in Italian Cypress, Cupressus sempervirens, in Response to Infection by the Fungus Seiridium cardinale.

Seiridium cardinale, the main fungal pathogen responsible for cypress bark canker, is the largest threat to cypresses worldwide. The terpene response of canker-resistant clones of Italian cypress, Cupressus sempervirens, to two differently aggressive isolates of S. cardinale was studied. Phloem terpene concentrations, foliar terpene concentrations, as well as foliar terpene emission rates were analyzed 1, 10, 30, and 90 days after artificial inoculation with fungal isolates. The phloem surrounding the inoculation point exhibited de novo production of four oxygenated monoterpenes and two unidentified terpenes. The concentrations of several constitutive mono- and diterpenes increased strongly (especially α-thujene, sabinene, terpinolene, terpinen-4-ol, oxygenated monoterpenes, manool, and two unidentified diterpenes) as the infection progressed. The proportion of minor terpenes in the infected cypresses increased markedly from the first day after inoculation (from 10% in the control to 30-50% in the infected treatments). Foliar concentrations showed no clear trend, but emission rates peaked at day 10 in infected trees, with higher δ-3-carene (15-fold) and total monoterpene (10-fold) emissions than the control. No substantial differences were found among cypresses infected by the two fungal isolates. These results suggest that cypresses activate several direct and indirect chemical defense mechanisms after infection by S. cardinale.

"Microscopic evidences of heavy metals distribution and anatomic alterations in breaching-leaves of Cupressus lindleyi growing around mining wastes".

In this article a study of the distribution of heavy metals in Cupressus lindleyi breaching-leaves was done in Taxco, Guerrero. At the same, heavy metals micro-localization was conducted in the breaching-leaves to understand the structural changes provoked by mining waste on plants. The most abundant contaminants in soils, tailings and different plant organs (roots, stems, and leaves) were Zn, Mn, and Pb. Nevertheless, As was more accumulated in the stem and breaching-leaves. The translocation factor and the bio-concentration factor were less than 1. The structural changes observed were the great accumulation of starch grains and phenolic compounds in the palisade parenchyma, changes in the hypodermis cell wall and necrotic zones in the palisade parenchyma. The distribution of heavy metals in breaching-leaves tissues was homogeneous in most of the elements. These results showed that C. lindleyi is a species that can be employed in phytostabilization of contaminated zones with mining waste because it is a native plant that does not require a lot of conditions for its development.

A novel synthetic pathway for tropolone ring formation via the olefin monoterpene intermediate terpinolene in cultured Cupressus lusitanica cells.

ß-Thujaplicin is a wood monoterpene and tropolone compound with a unique conjugated 7-membered ring. Because of its strong antifungal and antitumor activities, ß-thujaplicin is used in several fields. The biosynthesis pathway of ß-thujaplicin has not yet been elucidated. Using Cupressus lusitanica cell cultures in a radioisotope feeding experiment, our group previously demonstrated that geranyl pyrophosphate (GPP) is the starting material of ß-thujaplicin biosynthesis. The results of our previous terpene synthase assay suggested that terpinolene is the first olefin terpenoid intermediate from GPP to ß-thujaplicin, although there was no experimental evidence of this at that time. In the present study, we fed deuterium-labeled terpinolene to cultured C. lusitanica cells to determine whether terpinolene is an intermediate metabolite of ß-thujaplicin biosynthesis. A gas chromatography-mass spectroscopy analysis of the cell extracts from labeled terpinolene cultures revealed a peak of labeled ß-thujaplicin that was not observed after treatment with non-labeled terpinolene. The identification of labeled ß-thujaplicin was also performed by mass spectrum assignment. The outcome indicated that terpinolene is indeed an intermediate metabolite of ß-thujaplicin biosynthesis. To the best of our knowledge, there has been no prior report that tropolone compounds are biosynthesized via a terpene biosynthesis system, and our results thus suggest the existence of a novel biosynthetic pathway that produces the conjugated 7-membered ring.

Seasonal changes in copper and cobalt concentrations of Pinus nigra L., Cedrus libani and Cupressus arizonica leaves to monitor the effects of pollution in Elazig (Turkey).

The aim of this study is to examine seasonal changes in Cu and Co concentrations of three plant species for monitoring the effects of pollution in Elazig, Turkey. For this purpose, the leaves of the Pinus nigra L., Cedrus libani and Cupressus arizonica together with soil samples were collected from different points depending on traffic intensity, nearness the city center and cement factory as well as control location during different months of the year. Flame atomic absorption spectrophotometer (FAAS) was used for measurement of the metals in clear digests after the dry ashing method. Copper and Co concentrations were in the ranges from 1.3 to 2.6 mg x kg(-1) and < LOD to 0.26 mg x kg(-1) for Pinus nigra L., 1.2 to 4.7 mg x kg(-1) and < LOD to 0.41 mg x kg(-1) for Cedrus libani and 1.5 to 4.8 mg x kg(-1) and < LOD to 0.42 mg x kg(-1) for Cupressus arizonica, respectively. The levels observed for Cu and Co in the soil ranged from 12 to 38 mg x kg(-1) and 6.0 to 17 mg x kg(-1), respectively.

Proton-dependent coniferin transport, a common major transport event in differentiating xylem tissue of woody plants.

Lignin biosynthesis is an essential physiological activity of vascular plants if they are to survive under various environmental stresses on land. The biosynthesis of lignin proceeds in the cell wall by polymerization of precursors; the initial step of lignin polymerization is the transportation of lignin monomers from the cytosol to the cell wall, which is critical for lignin formation. There has been much debate on the transported form of the lignin precursor, either as free monolignols or their glucosides. In this study, we performed biochemical analyses to characterize the membrane transport mechanism of lignin precursors using angiosperms, hybrid poplar (Populus sieboldii × Populus grandidentata) and poplar (Populus sieboldii), as well gymnosperms, Japanese cypress (Chamaecyparis obtusa) and pine (Pinus densiflora). Membrane vesicles prepared from differentiating xylem tissues showed clear ATP-dependent transport activity of coniferin, whereas less than 4% of the coniferin transport activity was seen for coniferyl alcohol. Bafilomycin A1 and proton gradient erasers markedly inhibited coniferin transport in hybrid poplar membrane vesicles; in contrast, vanadate had no effect. Cis-inhibition experiments suggested that this transport activity was specific for coniferin. Membrane fractionation of hybrid poplar microsomes demonstrated that transport activity was localized to the tonoplast- and endomembrane-rich fraction. Differentiating xylem of Japanese cypress exhibited almost identical transport properties, suggesting the involvement of a common endomembrane-associated proton/coniferin antiport mechanism in the lignifying tissues of woody plants, both angiosperms and gymnosperms.

Ethanol-based organosolv treatment with trace hydrochloric acid improves the enzymatic digestibility of Japanese cypress (Chamaecyparis obtusa) by exposing nanofibers on the surface.

The effects of adding trace acids in ethanol based organosolv treatment were investigated to increase the enzymatic digestibility of Japanese cypress. A high glucose yield (60%) in the enzymatic hydrolysis was obtained by treating the sample at 170 °C for 45 min in 50% ethanol liquor containing 0.4% hydrochloric acid. Moreover, the enzymatic digestibility of the treated sample was improved to ∼70% by changing the enzyme from acremonium cellulase to Accellerase1500. Field emission scanning electron microscopy revealed the presence of lignin droplets and partial cellulose nanofibers on the surface of the treated sample. Simultaneous saccharification and fermentation of the treated samples using thermotolerant yeast (Kluyveromyces marxianus NBRC1777) was tested. A high ethanol concentration (22.1 g/L) was achieved using the EtOH50/W50/HCl0.4-treated sample compared with samples from other treatments.

A rapid biosynthesis route for the preparation of gold nanoparticles by aqueous extract of cypress leaves at room temperature.

In the present study, green synthesis of gold nanoparticles was reported using the aqueous extract of cypress leaves. The reduction of gold salt with the extract of cypress leaves resulted in the formation of gold nanoparticles. Effects of extract concentration and extract pH were investigated on the size of the nanoparticles. It was found that the average particle size of synthesized gold nanoparticles depends strongly on extract concentration and extract pH. FT-IR spectroscopy showed that bioorganic capping molecules were bound to the surface of particles. X-ray techniques confirmed the formation of gold nanoparticles and their crystalline structure. The inductively coupled plasma atomic emission spectroscopy analysis displayed that the reaction progress is higher than 90% at room temperature. Gold nanoparticles were mostly spherical in shape along with some irregular shapes. Cypress is an evergreen plant and its leaves are easily available in all four seasons. Also, the rate of the reaction was high and it was completed in only 10min. For these reasons, this method is cost-effective and environmentally friendly. Thus, it can be used in the synthesis of gold nanoparticles instead of chemical methods and other biosynthesis approaches.

Insights into a hydration regulating system in Cupressus pollen grains.

BACKGROUND AND AIMS: Hydration, rupture and exine opening due to the sudden and large expansion of intine are typical of taxoid-type pollen grains. A hemispheric outgrowth external to the exine was observed on Cupressus and Juniperus pollen grains before the intine swelling and exine release. However, the actual existence of this permanent or temporary structure and its precise role in pollen hydration is still being debated. The aim of this paper is to collect information on the actual presence of this peculiar outgrowth on the surface of the Cupressus pollen grain, its structure, composition and function. METHODS: Pollen grains of several Cupressus species were observed using various techniques and methodologies, under light and fluorescence microscopy, phase-contrast microscopy, confocal microscopy, scanning electron microscopy, and an environmental scanning electron microscope. Observations were also performed on other species with taxoid-type pollen grains. KEY RESULTS: A temporary structure located just above the pore was observed on Cupressus pollen grains, as well as on other taxoid-type pollens. It is hemispheric, layered, and consists of polysaccharides and proteins. The latter are confined to its inner part. Its presence seems to regulate the entrance of water into the grains at the beginning of pollen hydration. CONCLUSIONS: The presence of a temporary structure over the pore of taxoid-type pollen grains was confirmed and its structure was resolved using several stains and observation techniques. This structure plays a role in the first phases of pollen hydration.

[Effects of drought stress and re-watering on the active oxygen scavenging system of Cupressus funebris seedlings in Karst area].

This paper studied the active oxygen scavenging system of Cupressus funebris seedlings under drought condition and the recovery capability of the system after re-watering, aimed to understand the adaptation mechanisms of C. funebris to the 'drought and re-watering' environment in Karst area. With the increasing time of drought stress, the seedling's relative water content (RWC) decreased, soluble protein concentration increased first and decreased then, and malondialdehyde (MDA) content increased consistently. The MDA content recovered to the level of the control (CK) when re-watering was implemented within 2 weeks of drought, but could not when the re-watering was made after 4 and 6 weeks of drought. Under drought stress, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) increased consistently. After rewatering, the SOD activity had somewhat decrease but still remained at a higher level than the CK, and the POD and CAT activities decreased to the CK level when suffered mild stress but had less decrement when suffered severe stress. It was concluded that C. funebris seedlings could resist mild drought stress via increasing their soluble protein concentration and inhibiting membrane lipid peroxidation, but could not resist severe drought stress because of the irreversible damage of their membrane structure.

Effectiveness of Cupressus sempervirens cones as biosorbent for the removal of basic dyes from aqueous solutions in batch and dynamic modes.

The feasibility of using cypress cone chips from Cupressus sempervirens as a low-cost biosorbent for the removal of two representative basic dyes, methylene blue (MB) and rhodamine B (RhB), from aqueous solutions was investigated in batch and continuous modes. Dyes biosorption was strongly dependent on the solution's pH. Sorption kinetics was determined and properly described by the pseudo-second-order rate model. Experimental equilibrium isotherms fitted the Langmuir model, showing maximum biosorption capacities of 0.62 mmol/g for MB and 0.24 mmol/g for RhB. Competitive experiments from a binary solution of the dyes demonstrated the preference of the cone chips for biosorbing MB. Very low desorption efficiencies were obtained for both dyes. Dynamic experiments showed that the breakthrough time was three times higher for MB biosorption than for RhB for the same conditions. Breakthrough curves were properly represented by a mathematical model.

A modified protocol for RNA isolation from high polysaccharide containing Cupressus arizonica pollen. Applications for RT-PCR and phage display library construction.

RNA isolation is the first step in the study of gene expression and recombinant protein production. However, the isolation of high quantity and high-quality RNA from tissues containing large amounts of polysaccharides has proven to be a difficult process. Cupressus arizonica pollen, in addition to containing high polysaccharide levels, is a challenging starting material for RNA isolation due to the roughness of the pollen grain's walls. Here, we describe an improved technique for RNA isolation from C. arizonica pollen grains. The protocol includes a special disruption and homogenization process as well as a two-step modified RNA isolation technique which consists of an acid phenol extraction followed by a final cleanup using a commercial kit. Resulting RNA proved to be free of contaminants as determined by UV spectrophotometry. The quality of the RNA was analyzed on a bioanalyzer and showed visible 25S and 18S bands. This RNA was successfully used in downstream applications such as RT-PCR and phage display library construction.

Volatile and non-volatile monoterpenes produced by elicitor-stimulated Cupressus lusitanica cultured cells.

Elicitor treatment initiates defense responses in cultured Cupressus lusitanica cells. In order to investigate the defense mechanism with a yeast extract elicitor, we carried out solid-phase microextraction coupled with gas chromatography for monoterpene analysis. Ten hydrocarbon monoterpenes, including high amounts of sabinene and limonene, were detected in the gas phase of the elicitor-treated cell cultures. Six oxidized monoterpenes including beta-thujaplicin were also detected in the ether extract of the cells and the medium. Time-course profiles of volatile monoterpenes showed that one group of hydrocarbon monoterpenes was maximized on the second day after elicitation, while the other group was maximized on the third day. There were no oxidized monoterpenes that are structurally related to sabinene and limonene in the gas phase or cell extracts, suggesting that these compounds are produced exclusively for emission. Other monoterpenes, which are produced during later stages of elicitation, are metabolized into more complex compounds such as oxidized monoterpenes, including beta-thujaplicin. Although terpinolene synthase was the principal monoterpene synthase in these cell cultures, terpinolene was detected only as a minor compound in the gas phase. The time course for terpinolene synthase activity coincided with beta-thujaplicin biosynthesis. Thus, most of the terpinolene is metabolized rapidly to oxidized terpenes such as beta-thujaplicin rather than emitted.