Widespread Occurrence of Non-Extractable Fluorine in Artificial Turfs from Stockholm, Sweden.

Per- and polyfluoroalkyl substances (PFAS) are frequently used in the production of rubber and plastic, but little is known about the identity, concentration, or prevalence of PFAS in these products. In this study, a representative sample of plastic- and rubber-containing artificial turf (AT) fields from Stockholm, Sweden, was subjected to total fluorine (TF), extractable organic fluorine (EOF), and target PFAS analysis. TF was observed in all 51 AT samples (ranges of 16-313, 12-310, and 24-661 µg of F/g in backing, filling, and blades, respectively), while EOF and target PFAS occurred in <42% of all samples (<200 and <1 ng of F/g, respectively). A subset of samples extracted with water confirmed the absence of fluoride. Moreover, application of the total oxidizable precursor assay revealed negligible perfluoroalkyl acid (PFAA) formation across all three sample types, indicating that the fluorinated substances in AT are not low-molecular weight PFAA precursors. Collectively, these results point toward polymeric organofluorine (e.g., fluoroelastomer, polytetrafluoroethylene, and polyvinylidene fluoride), consistent with patent literature. The combination of poor extractability and recalcitrance toward advanced oxidation suggests that the fluorine in AT does not pose an imminent risk to users. However, concerns surrounding the production and end of life of AT, as well as the contribution of filling and blades to environmental microplastic contamination, remain.

The effect of reduction measures on concentrations of hazardous semivolatile organic compounds in indoor air and dust of Swedish preschools.

Young children spend a substantial part of their waking time in preschools. It is therefore important to reduce the load of hazardous semivolatile organic compounds (SVOCs) in the preschools' indoor environment. The presence and levels of five SVOC groups were evaluated (1) in a newly built preschool, (2) before and after renovation of a preschool, and (3) in a preschool where SVOC-containing articles were removed. The new building and the renovation were performed using construction materials that were approved with respect to content of restricted chemicals. SVOC substance groups were measured in indoor air and settled dust and included phthalates and alternative plasticizers, organophosphate esters (OPEs), brominated flame retardants, and bisphenols. The most abundant substance groups in both indoor air and dust were phthalates and alternative plasticizers and OPEs. SVOC concentrations were lower or of the same order of magnitude as those reported in comparable studies. The relative Cumulative Hazard Quotient (HQcum ) was used to assess the effects of the different reduction measures on children's SVOC exposure from indoor air and dust in the preschools. HQcum values were low (1.0-6.1%) in all three preschools and decreased further after renovation and article substitution. The SVOCs concentrations decreased significantly more in the preschool renovated with the approved building materials than in the preschool where the SVOC-containing articles were removed.

Adhesion of Streptococcus mitis and Actinomyces oris in co-culture to machined and anodized titanium surfaces as affected by atmosphere and pH.

BACKGROUND: With the rising demand for osseointegrated titanium implants for replacing missing teeth, often in patients with a history of periodontitis, implant-related infections have become an issue of growing concern. Novel methods for treating and preventing implant-associated infections are urgently needed. The aim of this study was to investigate if different pH, atmosphere and surface properties could restrict bacterial adhesion to titanium surfaces used in dental implants. METHODS: Titanium discs with machined or anodized (TiUnite™) surface were incubated with a co-culture of Streptococcus mitis and Actinomyces oris (early colonizers of oral surfaces) at pH 5.0, 7.0 and 9.0 at aerobic or anaerobic atmosphere. The adhesion was analysed by counting colony forming (CFU) units on agar and by confocal laser scanning microscopy (CLSM). RESULTS: The CFU analysis showed that a pH of 5.0 was found to significantly decrease the adhesion of S. mitis, and an aerobic atmosphere, the adhesion of A. oris. S. mitis was found in significantly less amounts on the anodized surface than the machined surface, while A. oris was found in equal amounts on both surfaces. The CLSM analysis confirmed the results from the CFU count and provided additional information on how the two oral commensal species adhered to the surfaces: mainly in dispersed clusters oriented with the groves of the machined surface and the pores of the anodized surface. CONCLUSIONS: Bacterial adhesion by S. mitis and A. oris can be restricted by acidic pH and aerobic atmosphere. The anodized surface reduced the adhesion of S. mitis compared to the machined surface; while A. oris adhered equally well to the pores of the anodized surface and to the grooves of the machined surface. It is difficult to transfer these results directly into a clinical situation. However, it is worth further investigating these findings from an in vitro perspective, as well as clinically, to gain more knowledge of the effects acid pH and aerobic atmosphere have on initial bacterial adhesion.

Liver-derived IGF-I regulates mean life span in mice.

BACKGROUND: Transgenic mice with low levels of global insulin-like growth factor-I (IGF-I) throughout their life span, including pre- and postnatal development, have increased longevity. This study investigated whether specific deficiency of liver-derived, endocrine IGF-I is of importance for life span. METHODS AND FINDINGS: Serum IGF-I was reduced by approximately 80% in mice with adult, liver-specific IGF-I inactivation (LI-IGF-I(-/-) mice), and body weight decreased due to reduced body fat. The mean life span of LI-IGF-I(-/-) mice (n = 84) increased 10% vs. control mice (n = 137) (Cox's test, p<0.01), mainly due to increased life span (16%) of female mice [LI-IGF-I(-/-) mice (n = 31): 26.7±1.1 vs. control (n = 67): 23.0±0.7 months, p<0.001]. Male LI-IGF-I(-/-) mice showed only a tendency for increased longevity (p = 0.10). Energy expenditure, measured as oxygen consumption during and after submaximal exercise, was increased in the LI-IGF-I(-/-) mice. Moreover, microarray and RT-PCR analyses showed consistent regulation of three genes (heat shock protein 1A and 1B and connective tissue growth factor) in several body organs in the LI-IGF-I(-/-) mice. CONCLUSIONS: Adult inactivation of liver-derived, endocrine IGF-I resulted in moderately increased mean life span. Body weight and body fat decreased in LI-IGF-I(-/-) mice, possibly due to increased energy expenditure during exercise. Genes earlier reported to modulate stress response and collagen aging showed consistent regulation, providing mechanisms that could underlie the increased mean life span in the LI-IGF-I(-/-) mice.

Argon ion beam polishing: a preparation technique for evaluating the interface of osseointegrated implants with high resolution.

PURPOSE: The objective of this study was to assess the use of ion beam polishing for preparing cross sections suitable for high-resolution scanning electron microscope (SEM) investigation of dental implants with a brittle porous oxide layer and of bone/implant interfaces. MATERIALS AND METHODS: Thirteen Nobel Biocare TiUnite implants were placed in minipigs. After 4 weeks, the implant and surrounding bone were removed en bloc and the implant was cut axially into two halves. The cross section was then polished with an argon ion beam. Additionally, ion beam-polished cross sections were prepared from four as-received implants. Ion beam-polished surfaces were studied with a field emission SEM (FE-SEM). RESULTS: With FE-SEM, up to 1 mm along the interface of ion beam-polished implant surfaces can be studied with a resolution of a few nanometers. Filled and unfilled pores of the porous TiUnite coating can be distinguished, providing information on pore accessibility. Implant-bone interfaces can be analyzed using backscattered electron images, where titanium, the oxide layer, mineralized extracellular matrix, and osteocyte lacunae/resin/soft tissue can easily be distinguished as a result of atomic number contrast and the sharp boundaries between the different materials. Filled and unfilled pores can be distinguished. Characterization of local chemistry is possible with energy dispersive X-ray spectrometry, and bone growth into small pores (< 1 µm) can be unambiguously confirmed. CONCLUSION: FE-SEM complements the established methods for the characterization of interfaces and bridges the wide gap in accessible length scale and resolution between the observations of mechanically polished interfaces by optical or scanning electron microscopes and the observation of focused ion beam-milled sections in a transmission electron microscope.

Virtual variation simulation of CAD/CAM template-guided surgeries performed on human cadavers: Part II.

STATEMENT OF PROBLEM: CAD/CAM template-guided surgery has gained attention as a method of improving the predictability of dental implant placement. However, due to possible variations during the manufacturing process and in the robustness of the template design, a virtual prediction of the potential positioning of the implants is needed. PURPOSE: The purpose of this study was to perform virtual variation simulations on virtually planned implant placements and to compare them with corresponding results from actual surgeries performed on human cadavers in a previous study. MATERIAL AND METHODS: Seventeen computer-aided plans were used for virtual variation simulation of surgeries conducted on 17 human cadavers and 145 implants placed in the cadavers. For each surgery, 10,000 virtual surgeries were performed, resulting in 1,450,000 implant placements. The results from the virtual variation simulations were statistically compared with the results from the actual surgeries. The Mann-Whitney U test was used to compare the implant distributions (alpha=.05). RESULTS: In the maxillae, the difference between the simulated average mean of the mean and the compared surgical average of the median was 0.22 mm (apex) and -0.35 mm (hex), and for the mandible, the corresponding values were -0.19 mm (apex) and -0.69 mm (hex). The simulated average mean of the range compared to the mean range of the maximum deviation results from the surgeries of the maxillae was 2.96 mm (apex) and 0.44 mm (hex), and 2.3 mm (apex) and 0.26 mm (hex) for the mandible. The implant distributions between the simulations and the surgeries were significantly different at both the hex (P<.001) and apex (P<.001). CONCLUSIONS: The implant distributions were neither static nor normally distributed. Thus, within the limitations of this study, the definitive geometrical variations of the implants were not static, as they depend on the individual anatomy of the jaws and the ability to place the CAD/CAM-guided surgical template in the proper position.

Accuracy of CAD/CAM-guided surgical template implant surgery on human cadavers: Part I.

STATEMENT OF PROBLEM: An optimal method for approaching the clinical surgical situation, when using preoperatively, virtually planned implant positioning, is to transfer data to a CAD/CAM-guided surgical template with the definitive position of the implant placed after surgery. The accuracy of CAD/CAM-guided surgeries must be determined to provide safe treatment. PURPOSE: The purpose of this study was to compare the deviation between the position of virtually planned implants and the position of implants placed with a CAD/CAM-guided surgical template in the mandible and the maxilla in human cadavers. MATERIAL AND METHODS: Ten maxillae and 7 mandibles, from completely edentulous cadavers, were scanned with CT, and 145 implants (Brånemark RP Groovy) were planned with software and placed with the aid of a CAD/CAM-guided surgical template. The preoperative CT scan was matched with the postoperative CT scan using voxel-based registration. The positions of the virtually planned implants were compared with the actual positions of the implants. Data were analyzed with a t test (alpha=.05). RESULTS: The mean measurement differences between the computer-planned implants and implants placed after surgery for all implants placed were 1.25 mm (95% CI: 1.13-1.36) for the apex, 1.06 mm (95% CI: 0.97-1.16) for the hex, 0.28 mm (95% CI: 0.18-0.38) for the depth deviation, 2.64 degrees (95% CI: 2.41-2.87) for the angular deviation, and 0.71 mm (95% CI: 0.61-0.81 mm) for the translation deviation. CONCLUSIONS: The results demonstrated a statistically significant difference between mandibles and maxillae for the hex, apex, and depth measurements in the variation between the virtually planned implant positions and the positions of the implants placed after surgery with a CAD/CAM-guided surgical template.

Influence of fungal infection and wounding on contents and enantiomeric compositions of monoterpenes in phloem of Pinus sylvestris.

To identify chemical resistant markers induced by fungal or mechanical injury, young trees of Scots pine (Pinus sylvestris) were subjected to inoculations of blue stain fungi associated with the pine shoot beetles Tomicus piniperda and T. minor. Among the 20 trees selected for chemical analyses, 16 were divided into four groups: one as control and three were pretreated by wounding only, or by inoculation with either the blue stain fungus Leptographium wingfieldii or Ophiostoma canum. Four wk after pretreatment, all 16 pretreated trees were mass-inoculated with L. wingfieldii. The absolute and relative amounts, as well as the enantiomeric compositions of monoterpene hydrocarbons in the phloem, were determined via a small sample of the phloem before and after the pretreatment and mass inoculation, by using two-dimensional gas chromatography (2D GC) and GC-mass spectrometry (MS). After mass inoculation, the absolute amounts of most of the monoterpenes decreased in the phloem sampled >20 cm from the fungal infection, and were higher in the phloem sampled within the infected reaction zone. The relative amounts of both (-)-beta-pinene and (-)-limonene increased in phloem samples taken >20 cm above the fungal inoculation in the preinoculated trees compared with phloem sampled from the remaining four control trees. The enantiomeric compositions of beta-pinene and limonene changed, after fungal growth, at defined distances from the inoculation site: the proportion of the (-)-enantiomers was highest in the phloem sampled >20 cm from the fungal inoculation. Four wk after pretreatment, monoterpene production in the phloem at the site of inoculation was more enhanced by L. wingfieldii than by O. canum. However, the different virulence levels of the fungi did not affect the enantiomeric composition of the monoterpenes. The biosynthesis of monoterpene enantiomers is discussed in relation to induced pathogen resistance.

Platelet-derived growth factor B retention is essential for development of normal structure and function of conduit vessels and capillaries.

OBJECTIVE: Extracellular retention of PDGF-B has been proposed to play an important role in PDGF-B signalling. We used the PDGF-B retention motif knockout mouse (RetKO) to study the effects of retention motif deletion on development of micro- and macrovascular structure and function. METHODS: Passive and active properties of conduit vessels were studied using myograph techniques and histological examination. Capillary structure and function was studied using measurements of capillary density in skeletal muscle and by assessing aerobic physical performance in a treadmill setup. Cardiac function was assessed using echocardiography. RESULTS: Myograph experiments revealed an increased diameter and stiffness of the aorta in RetKO. Histological examination showed increased media collagen content and a decreased number of aortic wall layers, however with a similar number of vascular smooth muscle cells. This outward eutrophic remodelling of the aorta was accompanied by endothelial dysfunction. RetKO showed decreased capillary density in skeletal muscle and signs of a defective delivery of capillary oxygen to skeletal muscle, as shown by a decreased physical performance. In RetKO mice, echocardiography revealed an adaptive eccentric cardiac hypertrophy. CONCLUSION: We conclude that retention of PDGF-B during development is essential for a normal conduit vessel function in the adult mouse. Furthermore, PDGF-B retention is also necessary for the development of an adequate capillary density, and thereby for a normal oxygen delivery to skeletal muscle. The lack of primary effects on cardiac function supports the redundant role of PDGF-B in cardiac development.

Host selection in Tomicus piniperda L.: composition of monoterpene hydrocarbons in relation to attack frequency in the shoot feeding phase.

The aim of this study was to investigate the host selection capacity of the pine shoot beetle, Tomicus piniperda, in the shoot-feeding phase and analyze the chiral and non-chiral host volatiles by means of GC-MS and 2D-GC in five Pinus species originating from France (Pinus sylvestris, P. halepensis, P. nigra laricio, P. pinaster maritima, P. pinaster mesogeensis). Dominating monoterpenes were (-)-alpha-pinene, (+)-alpha-pinene, (-)-beta-pinene and (+)-3-carene. The amounts of the enantiomers varied considerably within and among the species. In a principal component analysis-plot, based on the absolute amounts of 18 monoterpene hydrocarbons, separation of the pine species into two groups was obtained. P. halepensis and P. sylvestris were grouped according to the amount of (+)-alpha-pinene and (+)-3-carene, while P. nigra laricio, P. pinaster maritima and P. pinaster mesogeensis were grouped according to (-)-alpha-pinene and (-)-beta-pinene. P. nigra laricio was the species most attacked and P, halepensis the one least attacked by T. piniperda.

Reduced stress- and cold-induced increase in energy expenditure in interleukin-6-deficient mice.

Interleukin-6 (IL-6) deficient (-/-) mice develop mature onset obesity. Pharmacological studies have shown that IL-6 has direct lipolytic effects and when administered centrally increases sympathetic outflow. However, the metabolic functions of endogenous IL-6 are not fully elucidated. We aimed to investigate the effect of IL-6 deficiency with respect to cold exposure and cage-switch stress, that is, situations that normally increase sympathetic outflow. Energy metabolism, core temperature, heart rate, and activity were investigated in young preobese IL-6-/- mice by indirect calorimetry together with telemetry. Baseline measurements and the effect of cage-switch stress were investigated at thermoneutrality (30 degrees C) and at room temperature (20 degrees C). The effect of cold exposure was investigated at 4 degrees C. At 30 degrees C, the basal core temperature was 0.6 +/- 0.24 degrees C lower in IL-6-/- compared with wild-type mice, whereas the oxygen consumption did not differ significantly. The respiratory exchange ratio at 20 degrees C was significantly higher and the calculated fat utilization rate was lower in IL-6-/- mice. In response to cage-switch stress, the increase in oxygen consumption at both 30 and 20 degrees C was lower in IL-6-/- than in wild-type mice. The increase in heart rate was lower in IL-6-/- mice at 30 degrees C. At 4 degrees C, both the oxygen consumption and core temperature were lower in IL-6-/- compared with wild-type mice, suggesting a lower cold-induced thermogenesis in IL-6-/- mice. The present results indicate that endogenous IL-6 is of importance for stress- and cold-induced energy expenditure in mice.

Enzymatic properties of native and deglycosylated hybrid aspen (Populus tremulaxtremuloides) xyloglucan endotransglycosylase 16A expressed in Pichia pastoris.

The cDNA encoding a xyloglucan endotransglycosylase, PttXET16A, from hybrid aspen (Populus tremulaxtremuloides) has been isolated from an expressed sequence tag library and expressed in the methylotrophic yeast Pichia pastoris. Sequence analysis indicated a high degree of similarity with other proteins in the XTH (xyloglucan transglycosylase/hydrolase) gene subfamily of GH16 (glycoside hydrolase family 16). In addition to the conserved GH16 catalytic sequence motif, PttXET16A contains a conserved N-glycosylation site situated proximal to the predicted catalytic residues. MS analysis indicated that the recombinant PttXET16A expressed in P. pastoris is heterogeneous due to the presence of variable N-glycosylation and incomplete cleavage of the alpha-factor secretion signal peptide. Removal of the N-glycan by endoglycosidase H treatment did not influence the catalytic activity significantly. Similarly, site-directed mutagenesis of Asn93 to serine to remove the N-glycosylation site resulted in an enzyme which was comparable with the wild-type enzyme in specific activity and thermal stability but had clearly reduced solubility. Hydrolytic activity was detected neither in wild-type PttXET16A before or after enzymatic deglycosylation nor in PttXET16A N93S (Asn93-->Ser) mutant.

Functional characterization of nine Norway Spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily.

Constitutive and induced terpenoids are important defense compounds for many plants against potential herbivores and pathogens. In Norway spruce (Picea abies L. Karst), treatment with methyl jasmonate induces complex chemical and biochemical terpenoid defense responses associated with traumatic resin duct development in stems and volatile terpenoid emissions in needles. The cloning of (+)-3-carene synthase was the first step in characterizing this system at the molecular genetic level. Here we report the isolation and functional characterization of nine additional terpene synthase (TPS) cDNAs from Norway spruce. These cDNAs encode four monoterpene synthases, myrcene synthase, (-)-limonene synthase, (-)-alpha/beta-pinene synthase, and (-)-linalool synthase; three sesquiterpene synthases, longifolene synthase, E,E-alpha-farnesene synthase, and E-alpha-bisabolene synthase; and two diterpene synthases, isopimara-7,15-diene synthase and levopimaradiene/abietadiene synthase, each with a unique product profile. To our knowledge, genes encoding isopimara-7,15-diene synthase and longifolene synthase have not been previously described, and this linalool synthase is the first described from a gymnosperm. These functionally diverse TPS account for much of the structural diversity of constitutive and methyl jasmonate-induced terpenoids in foliage, xylem, bark, and volatile emissions from needles of Norway spruce. Phylogenetic analyses based on the inclusion of these TPS into the TPS-d subfamily revealed that functional specialization of conifer TPS occurred before speciation of Pinaceae. Furthermore, based on TPS enclaves created by distinct branching patterns, the TPS-d subfamily is divided into three groups according to sequence similarities and functional assessment. Similarities of TPS evolution in angiosperms and modeling of TPS protein structures are discussed.

Reduced exercise endurance in interleukin-6-deficient mice.

IL-6 is produced and released in large amounts from skeletal muscle during prolonged exercise in both mice and humans, but there are few data indicating the biological significance of this. IL-6 exerts metabolic effects such as stimulating energy expenditure and reducing body fat mass. We have now investigated the effects of IL-6 deficiency on exercise endurance and energy expenditure in preobese and obese IL-6-deficient (IL-6(-/-)) mice. Four-month-old preobese and 7-month-old obese IL-6(-/-) male mice backcrossed to C57BL/6 and their littermate controls were exercised on a treadmill, and energy expenditure was measured as oxygen consumption with the use of indirect calorimetry. The preobese IL-6(-/-) mice were significantly leaner than the control mice, whereas the older IL-6(-/-) mice, as expected, had developed obesity. Resting young, but not older, IL-6(-/-) mice had an elevated respiratory exchange ratio (RER), indicating that they oxidize carbohydrates rather than fat for energy utilization. During exercise, the young and older IL-6(-/-) mice had a reduced endurance and a progressive decrease in oxygen consumption compared with control mice. There was no difference in RER in young IL-6(-/-) mice, whereas RER was enhanced in older IL-6(-/-), mice during exercise. In summary, IL-6(-/-) mice have reduced endurance and energy expenditure during exercise, suggesting that IL-6 is necessary for normal exercise capacity.

(E)-beta-ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: function and expression of three terpene synthase genes of a new terpene synthase subfamily.

Snapdragon flowers emit two monoterpene olefins, myrcene and (E)-beta-ocimene, derived from geranyl diphosphate, in addition to a major phenylpropanoid floral scent component, methylbenzoate. Emission of these monoterpenes is regulated developmentally and follows diurnal rhythms controlled by a circadian clock. Using a functional genomics approach, we have isolated and characterized three closely related cDNAs from a snapdragon petal-specific library that encode two myrcene synthases (ama1e20 and ama0c15) and an (E)-beta-ocimene synthase (ama0a23). Although the two myrcene synthases are almost identical (98%), except for the N-terminal 13 amino acids, and are catalytically active, yielding a single monoterpene product, myrcene, only ama0c15 is expressed at a high level in flowers and contributes to floral myrcene emission. (E)-beta-Ocimene synthase is highly similar to snapdragon myrcene synthases (92% amino acid identity) and produces predominantly (E)-beta-ocimene (97% of total monoterpene olefin product) with small amounts of (Z)-beta-ocimene and myrcene. These newly isolated snapdragon monoterpene synthases, together with Arabidopsis AtTPS14 (At1g61680), define a new subfamily of the terpene synthase (TPS) family designated the Tps-g group. Members of this new Tps-g group lack the RRx(8)W motif, which is a characteristic feature of the Tps-d and Tps-b monoterpene synthases, suggesting that the reaction mechanism of Tps-g monoterpene synthase product formation does not proceed via an RR-dependent isomerization of geranyl diphosphate to 3S-linalyl diphosphate, as shown previously for limonene cyclase. Analyses of tissue-specific, developmental, and rhythmic expression of these monoterpene synthase genes in snapdragon flowers revealed coordinated regulation of phenylpropanoid and isoprenoid scent production.

Functional identification of AtTPS03 as (E)-beta-ocimene synthase: a monoterpene synthase catalyzing jasmonate- and wound-induced volatile formation in Arabidopsis thaliana.

(E)-beta-Ocimene is one of the most commonly found monoterpenes of the volatile blends that are emitted from leaves in response to damage by herbivores or mechanical wounding. (E)-beta-Ocimene is also a component of many floral scents. Airborne (E)-beta-ocimene emitted from plants can serve as a chemical cue for the attraction of parasitoids or predators of plant herbivores and also as an attractant for pollinating insects. Furthermore, exposure of plants to (E)-beta-ocimene can activate defense gene expression. In this paper, we describe cDNA cloning and functional characterization of a gene encoding a highly specialized (E)-beta-ocimene synthase, AtTPS03, from Arabidopsis thaliana (L.) Heynh. AtTPS03 was identified as a member of the large AtTPS gene family of putative terpene synthases. A cDNA for AtTPS03 was expressed in Escherichia coli and the enzyme function identified in vitro. The A. thaliana (E)-beta-ocimene synthase produces almost exclusively (E)-beta-ocimene (94%) with minor amounts of the related acyclic monoterpenes (Z)-beta-ocimene (4%) and myrcene (2%). Transcripts for AtTPS03 were up-regulated in leaves of Arabidopsis in response to mechanical wounding and treatment with jasmonic acid, concurrent with induced emission of (E)-beta-ocimene. AtTPS03 provides an important gene for probing plant-insect and possibly plant-plant interactions mediated by terpenoid volatiles.

Traumatic resin defense in Norway spruce (Picea abies): methyl jasmonate-induced terpene synthase gene expression, and cDNA cloning and functional characterization of (+)-3-carene synthase.

Picea abies (L.) Karst. (Norway spruce) employs constitutive and induced resin terpenoids as major chemical and physical defense-shields against insects and pathogens. In recent work, we showed that a suite of terpenoids, monoterpenoids and diterpenoids was induced in stems of Norway spruce after treatment of trees with methyl jasmonate (MeJA) (Martin et al., 2002). Increase of enzyme activities of terpenoid biosynthesis and accumulation of terpenoids was associated with MeJA-induced de novo differentiation of xylem resin ducts. The formation of defense-related traumatic resin ducts was also found in Norway spruce after attack by stem boring insects or after infestation with fungal pathogens. In the present study, we analyzed the traumatic resin response in Norway spruce further at the molecular genetic level. Treatment of trees with MeJA induced transient transcript accumulation of monoterpenoid synthases and diterpenoid synthases in stem tissues of Norway spruce. In screening for defense-related terpenoid synthase (TPS) genes from Norway spruce, a full-length monoterpenoid synthase cDNA, PaJF67, was isolated and the recombinant enzyme expressed in E. coli and functionally characterized in vitro. The cloned PaJF67 cDNA represents a new monoterpenoid synthase gene and the gene product was identified as 3-carene synthase. The enzyme encoded by PaJF67 forms stereospecifically (+)-3-carene (78% of total product) together with minor acyclic and cyclic monoterpenes, including the mechanistically closely related terpinolene (11% of total product). (+)-3-Carene is a characteristic monoterpene of constitutive and induced oleoresin defense of Norway spruce and other members of the Pinaceae.