Ability of plant callus cultures to synthesize and accumulate lower terpenoids

Callus cultures derived from seven oil-producing plants have been maintained under different regimes of media, temperature and illumination, and have been assayed for ability both to synthesize and to accumulate mono- and sesqui-terpenes. Callus of Pinus radiata (the sole gymnosperm) accumulated (α- and β-pinenes at levels comparable with those in the parent stem and needles; and that of Jasminum officinale accumulated traces of several monoterpenes (< 0.1 % the amount in petals) but cultures of Rosmarinus officinalis, Lavandula angustifolia, Anethum graveolens, Ocimum basilicum and Tanacetum vulgare did not detectably accumulate the lower terpenoids or secrete them into the medium. However, all seven culture lines yielded cell-free extracts containing prenyltransferase and an isomerizing system (as assayed by conversion of IPP into GPP, NPP and FPP) with activities some fold greater than those extracted from the parent mature plants, or up to 90-fold the levels extractable from young seedlings of the various species. Callus of five of the species (A. graveolens and O. basilicum were not assayed) also contained MVA-kinase, MVAP-kinase, MVAPP-decarboxylase and IPP-DMAPP isomerase at levels comparable with those in the parent tissue. Hence the angiosperms yielded cultures that presumably contained the crucial enzyniic machinery necessary for the synthesis of the lower terpenoids, although accumulation of those compounds did not occur. Reasons for this unexpected situation are discussed. These results imply that callus cultures may be a convenient source of biomass for studies on the enzymes of terpenoid biosynthesis.     

Isolation and expression of cytochrome P450 genes in the antennae and gut of pine beetle Dendroctonus rhizophagus (Curculionidae: Scolytinae) following exposure to host monoterpenes

Bark beetles oxidize the defensive monoterpenes of their host trees both to detoxify them and convert them into components of their pheromone system. This oxidation is catalyzed by cytochrome P450 enzymes and occurs in different tissues of the insect, including the gut (i.e., the site where the beetle's pheromones are produced and accumulated) and the antennae (i.e., the olfactory organs used for perception of airborne defensive monoterpenes as well as other host-associated compounds and pheromones). We identified ten new CYP genes in the pine beetle Dendroctonus rhizophagus in either antennae or gut tissue after stimulation with the vapors of major host monoterpenes α-pinene, β-pinene and 3-carene. Five genes belong to the CYP4 family, four to the CYP6 family and one to the CYP9 family. Differential expression of almost all of the CYP genes was observed between sexes, and within these significant differences among time, stimuli, anatomical region, and their interactions were found upon exposure to host monoterpenes. Increased expression of cytochrome P450 genes suggests that they play a role in the detoxification of monoterpenes released by this insect's host trees.     

Essential oil yield,composition, bioactivity and leaf morphology of Juniperus oxycedrus L. from Bulgaria and Serbia

Juniperus oxycedrus L. (Cupressaceae Bartlett) is widely distributed in countries with a Mediterranean climate. All plant parts contain highly aromatic essential oil (EO) and recently there have been efforts to introduce it as a cultivated crop. The species is known for its large morphological and chemical variation and its debatable taxonomic status. This study aimed to (1) compare content, composition, and antimicrobial activity of J. oxycedrus EO samples from plants growing in Bulgaria and Serbia, and (2) quantify morphological variations of leaves. Тhe EO content (yield) in dried juniper leaves varied from 0.06% (Кopaonik, Serbia) to 0.24% (Markovo, Bulgaria). We identified 51 EO constituents, belonging to monoterpenes, sesquiterpenes, and diterpenes. The class monoterpenes (monoterpene hydrocarbons and oxygenated monoterpenes) were the predominant compounds, representing 38.6–65.4% of the total EO, consisting primarily of α-pinene, limonene, sabinene, β-pinene, and β-myrcene. In addition, α-pinene was the major oil constituent in plants from all locations. Sesquiterpenes (sesquiterpene hydrocarbons and oxygenated sesquiterpenes) were the second largest class of constituents, which represented 19.3% tо 33.6% of the total EO. γ-Elemene was found only in the EO of J. oxycedrus from Bulgaria, while a high concentration of α-curcumene was found only in samples from Serbia (7.5–7.8%). Significant differences in antimicrobial activity of the EO were found in bacterial strains Bacillus cereus and Streptococcus pneumoniae. There was no significant difference among the mean leaf width of the six combinations location x sex, and the overall leaf mean width was 1.24 mm. However, there was a significant difference between the mean leaf lengths. In this study, none of the studied populations had a higher concentration of limonene than of α-pinene, indicating that the flora of the two countries include J. oxycedrus and not the previously reported J. deltoides. The results revealed significant variation in EO profile that may contribute to the development of new cultivars of J. oxycedrus.     

Role of acyclic compounds in monoterpene biosynthesis in Pinus pinaster

The biosynthesis of monoterpene hydrocarbons was studied in maritime pine needles by incorporation of ¹⁴CO₂. It was shown that the acyclic terpenes β-myrcene and trans-β-ocimene, act as transitory compounds before the biosynthesis of cyclic monoterpenes such as α- and β-pinene. The mechanisms of biosynthesis are genetically controlled.     

Chemical composition of the cortical essential oil from Abies balsamea

Monoterpenoids and sesquiterpene hydrocarbons of Abies balsamea cortical oleoresin (Canada balsam) were analyzed by a combination of chromatographic and spectroscopic methods. Monoterpene hydrocarbons (21%) were composed of β-pinene, α-pinene, β-phellandrene, limonene, 3-carene, myrcene and camphene (listed in order of decreasing percentages), and oxygenated monoterpenes (0·4%) contained 4,4-dimethyl-2-cyclohepten-1-one, linalool, bornyl acetate, methylthymol, citronellyl acetate, α-terpineol, piperitone, citronellal, borneol, citronellol, two unknowns, and geraniol. From the sesquiterpene hydrocarbon fraction (1·1%) were isolated: longifolene, β-bisabolene, longipinene, an unknown, sativene, cyclosativene, cis-α-bisabolene, β-himachalene, α-himachalene, β-caryophyllene, γ-humulene, farnesene, longicyclene, an unknown, and β-selinene. Both himachalenes have been identified for the first time in Pinaceae outside of Cedrus; their co-occurrence with γ-humulene, longifolene, longipinene and longicyclene supports the biosynthetic mechanism by which all of these compounds arise through initial 1/11 cyclization of tran-cis-farnesylphosphate.     

Biosynthesis of monoterpenes by Ceratocystis moniliformis

Ceratocystis moniliformis produced and excreted monoterpenes when grown on potato-dextrose broth. Geraniol, nerol, citronellol, linalol, α-terpineol, geranial and neral were identified by GC-MS. Their production commenced with the depletion of nitrogen in the growth medium and their combined concentration peaked at about 50 μg/ml on the 5th day of growth. The pathway for the biosynthesis of the identified monoterpenes was studied by supplying the radioactive precursors mevalonic acid-[2-¹⁴C], l-leucine-[4,5-³H(N)], and acetate- [2-¹⁴C] to C. moniliformis. For each precursor, the extent of incorporation into the above monoterpenes and the distribution of radioactivity in geraniol was determined. It was concluded that monoterpenes were formed via the mevalonate pathway, previously established for higher terpenes in other organisms. This represents the first information available on the biosynthetic pathway for free monoterpenes in a microbial system.     

Isolation and characterization of terpene synthases in cotton (Gossypium hirsutum)

Cotton plants accumulate gossypol and related sesquiterpene aldehydes, which function as phytoalexins against pathogens and feeding deterrents to herbivorous insects. However, to date little is known about the biosynthesis of volatile terpenes in this crop. Herein is reported that 5 monoterpenes and 11 sesquiterpenes from extracts of a glanded cotton cultivar, Gossypium hirsutum cv. CCRI12, were detected by gas chromatography–mass spectrometry (GC–MS). By EST data mining combined with Rapid Amplification of cDNA Ends (RACE), full-length cDNAs of three terpene synthases (TPSs), GhTPS1, GhTPS2 and GhTPS3 were isolated. By in vitro assays of the recombinant proteins, it was found that GhTPS1 and GhTPS2 are sesquiterpene synthases: the former converted farnesyl pyrophosphate (FPP) into β-caryophyllene and α-humulene in a ratio of 2:1, whereas the latter produced several sesquiterpenes with guaia-1(10),11-diene as the major product. By contrast, GhTPS3 is a monoterpene synthase, which produced α-pinene, β-pinene, β-phellandrene and trace amounts of other monoterpenes from geranyl pyrophosphate (GPP). The TPS activities were also supported by Virus Induced Gene Silencing (VIGS) in the cotton plant. GhTPS1 and GhTPS3 were highly expressed in the cotton plant overall, whereas GhTPS2 was expressed only in leaves. When stimulated by mechanical wounding, Verticillium dahliae (Vde) elicitor or methyl jasmonate (MeJA), production of terpenes and expression of the corresponding synthase genes were induced. These data demonstrate that the three genes account for the biosynthesis of volatile terpenes of cotton, at least of this Upland cotton.     

Airborne limonene confers limited thermotolerance to Quercus ilex

The purpose of the study was to test the possible and controversial thermotolerance role of monoterpene production and emission and the related responses of antioxidants. Quercus ilex seedlings were exposed to a ramp of temperatures of 5°C steps from 25 to 50°C growing with and without limonene fumigation (7.5 µl l⁻¹). Net photosynthetic rates, maximal photochemical efficiency of PSII (F_v/F_m), oxidation state of ascorbic acid, and lipid peroxidation estimated by malondialdehyde concentrations of limonene-fumigated (LF) plants did not significantly differ from control (C) plants. No consistent changes in emissions of the other monoterpenes, α-pinene, β-phellandrene, β-pinene or β-myrcene were found. However, slight differences were found in the concentration of antioxidants. The amounts of α-tocopherol did not change or even tended to decrease at high temperatures in LF plants whereas they tended to increase by approximately 60% at 45 and 50°C relative to 25°C in C plants. Ascorbic acid reached its maximum concentration only at 45°C in LF plants whereas it reached its maximum at 35°C in C plants. β-Carotene did not decrease at high temperatures in LF plants whereas it decreased by approximately 15% at 45–50°C in C plants. Brown pigment index (BPI), an optical indicator of tissue oxidative processes, was lower in LF plants than in C plants. The photochemical reflectance index (PRI), an optical indicator of photosynthetic light use efficiency, was higher for LF plants than for C plants at elevated temperatures. Visual leaf damage (browning) tended to be less in LF plants than in C plans although not significantly (26.5 ± 8.5 versus 16.2 ± 4.8%). These results show that limonene does not confer clear and strong thermotolerance but might have some minor role. These results are in agreement with previous indications of weaker thermotolerance effect of monoterpenes than of isoprene.     

Microbial biotransformation of some monoterpene hydrocarbons

High commercial value compounds can be obtained through the microbial biotransformation of monoterpenes. Some of these monoterpenic substances are not expensive and produced in a variety of plant species. Biotransformation of some monoterpene hydrocarbons such as α-pinene, β-pinene, myrcene and p-cymene by 7 strain bacteria and 2 strain fungi was investigated. It was observed that some of microorganisms transformed monoterpenes to oxygenated monoterpenes in a good yield which among themStaphylococcus epidermidis showed higher yields.     

Characteristic alatoid ‘cineole cassette’ monoterpene synthase present in Nicotiana noctiflora

Nicotiana species of the section Alatae emit a characteristic floral scent comprising the? cineole cassette’ monoterpenes 1,8-cineole, limonene, myrcene, β-pinene, α-pinene, sabinene and α-terpineol. All previously isolated ‘cineole cassette’-monoterpene synthase genes are multi product enzymes that synthesize the seven compounds of the ‘cineole cassette’. Interestingly, so far this ‘alatoid’ trait was only shared with the eponymous species Nicotiana suaveolens of the sister section Suaveolentes. To determine the origin of the ‘cineole cassette’ monoterpene phenotype other potential parent species of section Noctiflorae or Petunoides as well as of the distantly related section Trigonophyllae were analysed. A monoterpene synthase producing the set of ‘cineole cassette’ compounds was isolated from N. noctiflorae. N. obtusifolia emitted solely 1,8-cineole and no monoterpenes were found in floral scents of N. petunoides and N. palmeri. Interestingly, the phylogenetic analysis clustered the new gene of N. noctiflora closely to the terpineol synthase genes of e.g. N. alata rather than to cineole synthase genes of e.g. N. forgetiana.     

Biosynthesis of geraniol and nerol in cell-free extracts of Tanacetum vulgare

Cell-free extracts from leaves of Tanacetum vulgare synthesised geraniol and nerol (3,7-dimethylocta-trans-2-ene-1-ol and its cis isomer) in up to 11·9 and 2·4% total yields from IPP-[4-¹⁴C] and MVA-[2-¹⁴C] respectively. Optimum preparations were obtained from plant material just before the onset of flowering. The ratio of the monoterpenols varied 28-fold for different preparations under conditions where these products or their phosphate esters were not interconverted. Similar extracts incorporated α-terpineol-[¹⁴C] and terpinen-4-ol-[¹⁴C] (p-menth-1-en-8- and -4-ol respectively) in 0·05 to 2·2% yields into a compound tentatively identified as isothujone (trans-thujan-3-one), and preparations from flowerheads converted IPP-[4-¹⁴C] in 2·7% yield into geranyl and neryl β-d-glucosides. Inhibitors of IPP-isomerase had little effect on the incorporation of IPP into the monoterpenols in cell-free systems from which endogenous compounds of low molecular-weight had been removed. The inference that a pool of protein-bonded DMAPP or its biogenetic equivalent was present was supported by the demonstration that geraniol and nerol biosynthesised in the absence of the inhibitors were predominantly (65 to 100%) labelled in the moiety derived from IPP.     

Callus in Rosmarinus officinalis L. (Lamiaceae): A morphoanatomical,histochemical and volatile analysis

This study aims to evaluate the anatomy of calli and their chemico-morphological characteristics after in vitro culture. Callus was induced from leaf explants of Rosmarinus officinalis derived from in vitro grown plants on media containing 2,4-dichlorophenoxyacetic acid (2,4D) and thidiazuron (TDZ) supplementation. Both capitate and peltate glandular trichomes were recognized in R. officinalis callus by light and electron microscopy. Histochemical tests showed positive reactions to lipophilic and terpenoid compounds for capitate trichomes and parenchyma or meristematic cells. Volatiles were isolated by simultaneous distillation–extraction and analysed by gas chromatography. Specifically, the use of 2,4D was shown to have a positive effect on the capacity of R. officinalis friable callus to produce volatile monoterpene compounds relative to TDZ. The main volatiles produced by callus were α-pinene, β-pinene and camphor produced in media containing 0.5 and 1.0 mg L^? 1 of 2,4D. Significant quantities of α-pinene, β-pinene and camphor were produced with 0.5 mg L^? 1 of 2,4D. On the basis of this accumulated evidence, it can be concluded that R. officinalis callus produces volatiles in developing glandular trichomes and that cellular aggregates and in vitro culture can be used for the production of volatiles under in vitro controlled conditions from selected plant material.     

Influence of environmental factors on content and composition of essential oil from common juniper ripe berry cones (Juniperus communis L.)

Abstract

The present study evaluated the influence of some environmental factors on the quantity and composition of essential oil (EO) in ripe berry cones of Juniperus communis L. The berry cones were collected from juniper shrubs growing wild at five localities of north-east Slovakia during the years 2012–2014. The EO yield ranged from 0.4 to 1.9%, depending on the locality and year. In the EO, eight monoterpenes (α-pinene, β-pinene, β-myrcene, sabinene, limonene, terpinene-4-ol, borneol, bornylacetate) and one sesquiterpene (β-caryophyllene) were identified. The dominant component was the monoterpene α-pinene, ranging from 31.0 to 49.0%. The amount and composition of the EO was affected by soil composition (content of humus and pH) and topographic environmental factors, including air temperature and precipitation. According to the composition of the EO, the studied juniper shrubs belong to the α-pinene chemotype.     

Chemical potential of Aphelandra sp. cell cultures

Six different callus lines and three different suspension culture lines were established from plants of two Aphelandra species (Acanthaceae). All established lines were analyzed for secondary metabolite accumulation. A discrepancy between secondary metabolites accumulated in the plants and in the cell cultures could be observed. All established Aphelandrasp. cell cultures produced verbascoside (acteoside) as the major extractable metabolite. Time course experiments were carried out to investigate the relationship between cell growth and verbascoside production. In the present study it was shown that verbascoside accumulation was growth dependent and positively related to the presence of 2,4-D in the medium. The conditions in which verbascoside represents ca. 18% of cell culture weight have been defined. Free polyamines were detected in the cell culture lines cultivated in MS liquid medium (cysteine 10 mg l^-1, thiamine 1 mg l^-1, 2,4-D 1 mg l^-1, kinetin 0.2 mg l^-1 and sucrose 30 g l^-1). Putrescine and spermidine accumulated within 8 days to a maximum of 8.4 μmol g^-1 of dry wt and 2.6 μmol g^-1 of dry wt respectively and thereafter their concentration decreased rapidly. There was no evidence for the presence of spermine or any other type of free or conjugated polyamines in the tested cell culture lines. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biotransformation of α- and β-pinene into flavor compounds

Products that bear the label “natural” have gained more attention in the marketplace. In this approach, the production of aroma compounds through biotransformation or bioconversion has been receiving more incentives in economic and research fields. Among the substrates used in these processes, terpenes can be highlighted for their versatility and low cost; some examples are limonene, α-pinene, and β-pinene. This work focused on the biotransformation of the two bicyclic monoterpenes, α-pinene and β-pinene; the use of different biocatalysts; the products obtained; and the conditions employed in the process.

     

油松萜烯成分变化与红脂大小蠹的反应特性

采用顶空采样方法,比较健康油松、人工损伤油松以及抗性油松在单萜烯成分组成上的差异。GC-MS分析表明,在自然状况下,油松树干释放的萜烯类成分很少,以α-蒎烯占绝对优势(>97%);人工损伤后,油松萜烯类成分明显增多,除α-蒎烯为主要成分外,还包括β-蒎烯、月桂烯、柠檬烯、萜品油烯、β-水芹烯、长叶烯等;而抗性油松萜烯类成分更为复杂。对3类油松主要单萜类成分的相对含量方差分析表明,α-蒎烯的相对含量呈显著降低;3-蒈烯在损伤寄主中相对含量最高,在抗性寄主中相对含量与自然状态下没有差异。柠檬烯、莰烯、萜品油烯在抗性寄主中相对比率显著增加。而β-蒎烯、月桂烯、β-水芹烯相对含量在3个处理中变化不大。在此基础上,比较红脂大小蠹Dendroctonus valens LeConte对油松主要单萜类成分的触角电位及嗅觉行为反应。结果表明,室内触角电位、嗅觉试验与先前林间试验结果相一致,即红脂大小蠹对(+)-3-蒈烯表现出最强的电生理和行为反应。对R-(+)-α-蒎烯和S-(-)-α-蒎烯研究发现,红脂大小蠹对α-蒎烯2个对映体的触角电位、嗅觉行为无显著不同。结合油松单萜类含量变化特点与红脂大小蠹行为反应,认为3-蒈烯相对含量上升可能作为易感寄主特点;而柠檬烯、莰烯、萜品油烯相对比率增加则代表了抗性或者非适合寄主的特征。     

Monoterpene differentiation in relation to the morphology of Pinus culminicola,Pinus nelsonii,Pinus pinceana and Pinus maximartinezii

Wood from 92 trees of Pinus culminicola, Pinus nelsonii, Pinus pinceana and Pinus maximartinezii from nine localities and oleoresin from several trees of Pinus nelsonii from one Iocality were analyzed for monoterpenes by gas liquid chromatography. Monoterpenes of Pinus nelsonii were composed mainly of α- and β-pinenes, while the main constituent of the monoterpenes of Pinus pinceana and Pinus maximartinezii was limonene, present in amounts of about 90 percent. The similarity in composition of the monoterpenes of Pinus pinceana and Pinus maximartinezii strongly supports the contention of Rzedowski [12] of the close evolutionary relation of the two pines. Pinus culminicola wood produced monoterpenes composed mainly of α-pinene, sabinene, terpinolene and limonene, with other monoterpenes occurring sporadically in sizeabie amounts. The variability between individual populations was relatively high, probably due to their long-time isolation by the intervening low-elevation barriers. Appreciable differences were found in compositions of monoterpenes from stem and from twigs of Pinus culrninicola.     

Bioactivity of essential oil of Litsea cubeba from China and its main compounds against two stored product insects

During our screening program for agrochemicals from Chinese medicinal herbs and wild plants, the essential oil of Litsea cubeba fruits was found to possess strong contact toxicity against the cigarette beetle Lasioderma serricorne adults and the booklouse Liposcelis bostrychophila, with LD₅₀ values of 27.33 μg/adult and 71.56 μg/cm², respectively, and also showed strong fumigant toxicity against the two stored product insects with LC₅₀ values of 22.97 and 0.73 mg/L, respectively. The essential oil obtained by hydrodistillation was investigated by GC MS. The main components of the essential oil were identified to be E-citral (geranial) (27.49%), Z-citral (neral) (23.57%) and d-limonene (18.82%) followed by β-thujene (3.34%), β-pinene (2.85%), α-pinene (2.57%), 6-methyl-5-hepten-2-one (2.40%) and linalool (2.36%). Citral (Z/E-citral), d-limonene, β-pinene, α-pinene and linalool were separated and purified by silica gel column chromatography and preparative thin layer chromatography, and further identified by means of physicochemical and spectrometric analysis. Citral and linalool showed strong contact toxicity against L. serricorne and L. bostrychophila (LD₅₀ = 11.76, 12.74 μg/adult and 20.15, 99.97 μg/cm², respectively) and fumigant toxicity against L. serricorne and L. bostrychophila (16.54, 18.04 mg/L air and 0.14, 0.71 mg/L air, respectively). Otherwise, citral, d-limonene and linalool were strongly repellent against the cigarette beetle L. serricorne as the essential oil whereas β-pinene and α-pinene exhibited weaker repellency against the cigarette beetle compared with the positive control, DEET. Moreover, except α-pinene and linalool, the other three compounds as well as the essential oil exhibited comparable repellency against the booklouse relative to DEET.     

Metabolism of linaloyl,neryl and geranyl pyrophosphates in Artemisia annua

The effectiveness of incorporation of radioactivity from ¹⁴C-labelled monoterpenyl pyrophosphates into isothujone, 1,8-cineol and α-pinene formed