A Novel Pathway for Sesquiterpene Biosynthesis from Z,Z-Farnesyl Pyrophosphate in the Wild Tomato Solanum habrochaites

In the wild tomato Solanum habrochaites, the Sst2 locus on chromosome 8 is responsible for the biosynthesis of several class II sesquiterpene olefins by glandular trichomes. Analysis of a trichome-specific EST collection from S. habrochaites revealed two candidate genes for the synthesis of Sst2-associated sesquiterpenes. zFPS encodes a protein with homology to Z-isoprenyl pyrophosphate synthases and SBS (for Santalene and Bergamotene Synthase) encodes a terpene synthase with homology to kaurene synthases. Both genes were found to cosegregate with the Sst2 locus. Recombinant zFPS protein catalyzed the synthesis of Z,Z-FPP from isopentenylpyrophosphate (IPP) and dimethylallylpyrophosphate (DMAPP), while coincubation of zFPS and SBS with the same substrates yielded a mixture of olefins identical to the Sst2-associated sesquiterpenes, including (+)-α-santalene, (+)-endo-β-bergamotene, and (−)-endo-α-bergamotene. In addition, headspace analysis of tobacco (Nicotiana sylvestris) plants expressing zFPS and SBS in glandular trichomes afforded the same mix of sesquiterpenes. Each of these proteins contains a putative plastid targeting sequence that mediates transport of a fused green fluorescent protein to the chloroplasts, suggesting that the biosynthesis of these sesquiterpenes uses IPP and DMAPP from the plastidic DXP pathway. These results provide novel insights into sesquiterpene biosynthesis and have general implications concerning sesquiterpene engineering in plants.     

Functional characterization of four sesquiterpene synthases from Ricinus communis (Castor bean)

Genome sequence analysis of Ricinus communis has indicated the presence of at least 22 putative terpene synthase (TPS) genes, 13 of which appear to encode sesquiterpene synthases (SeTPSs); however, no SeTPS genes have been isolated from this plant to date. cDNAs were recovered for six SeTPS candidates, and these were subjected to characterization in vivo and in vitro. The RcSeTPS candidates were expressed in either Escherichia coli or Saccharomyces cerevisiae strains with engineered sesquiterpene biosynthetic pathways, but only two (RcSeTPS1 and RcSeTPS7) produced detectable levels of product. In order to check whether the engineered microbial hosts were adequately engineered for sesquiterpene production, a selection of SeTPS genes was chosen from other plant species and demonstrated consistently high sesquiterpene titers. Activity could be demonstrated in vitro for two of the RcSeTPS candidates (RcSeTPS5 and RcSeTPS10) that were not observed to be functional in our microbial hosts. RcSeTPS1 produced two products, (−)-α-copaene and (+)-δ-cadinene, while RcSeTPS7 produced a single product, (E, E)-α-farnesene. Both RcSeTPS5 and RcSeTPS10 produced multiple sesquiterpenes.     

Within-plant distribution and emission of sesquiterpenes from Copaifera officinalis

Copaifera officinalis, the diesel tree, is known for massive production of oleoresin, mainly composed of sesquiterpene hydrocarbons. In this study, composition of these sesquiterpenes and their concentrations in leaves, stems and roots of C. officinalis at two developmental stages, including the three-week old (TW) seedlings and two-year old (TY) trees, were determined. The leaves of TW seedlings and TY trees contained similar number of sesquiterpenes, which also had comparable concentrations. The stems of TW seedlings had higher concentrations of sesquiterpenes than those of TY trees. In contrast, the number of sesquiterpene species and their concentrations in the roots of TW seedlings were much lower than those in the roots of TY trees. Cluster analysis of sesquiterpenes estimated that there are at least four terpene synthase genes involved in the production of sesquiterpenes in C. officinalis. Because sesquiterpenes are highly volatile, emissions of sesquiterpenes from healthy and wounded TW seedlings were examined using headspace analysis. Whereas very low emission of sesquiterpenes was detected from undamaged plants, the physically injured seedlings emitted a large number of sesquiterpenes, the quality and the relative quantity of which were similar to those in leaves determined using organic extraction. The implications of our findings to the biosynthetic pathways leading to the production of sesquiterpenes as well as their biological roles in C. officinalis are discussed.     

Essential oils from buds and leaves of two hazelnut (Corylus L.) cultivars with different resistance to filbert big bud mite (Phytoptus avellanae Nal.) and filbert aphid (Myzocallis coryli Goetze)

The aim of this study was to identify the allelopatic effect of the components of a mixture of essential oils (EO) contained in the buds and leaves of hazel (Corylus L.) on herbivores. We examined the effect of these compounds on the choice of plants of two different hazel cultivars by Phytoptus avellanae Nal. (filbert big bud mite) and Myzocallis coryli Goetze (filbert aphid), which are the most important pests of hazel in Poland and throughout the world. Our results show that plants of cv. ‘Mogulnus’ were more resistant than those of cv. ‘Barra’ to the feeding of mites and aphids in all study years. Using gas chromatography (GC) and GC/mass spectrometry methodology, we determined the qualitative and quantitative composition of EO in the buds and leaves of plants of these two hazel cultivars. The EO obtained from the analysed materials was a mixture of mono- and sesquiterpenes. The emission of volatile organic compounds from plants is known to repel or attract pests. The mixture of EO present in the hazel buds of cv. ‘Mogulus’, which is resistant to filbert big bud mite, was characterized by a high content of nerol, α-campholenol, methyl salicylate, spatulenol, β-caryophylene and δ-cadinene. In contrast, the leaves of this cultivar, colonized by filbert aphid but to a relatively small extent, contained greater quantities of nerol, α-campholenol, p-cymene, α-terpineol and germacrene D, than the leaves of cv. ‘Barra’, which is more accepted by aphids. However, the leaves of cv. ‘Barra’ were characterized by a considerably high content of menthol, limonene, isomenthone, methyl salicylate and L-menthone.     

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.     

Chemical composition and biological activities of Laurus essential oils from different Macaronesian Islands

Essential oils (EO) and eight pure components from the fresh leaves of Laurus novocanariensis (LN) from Madeira and of Laurus azorica (LA) from four Azorean Islands (S. Miguel-SMI, S. Maria-SMA, Pico-PIC and S. Jorge-SJO) were evaluated for fumigant and contact insecticidal effects on adult stage of Mediterranean fruit fly (medfly) and for inhibition of oviposition. The chemical composition of EO were analysed by GC/MS. Oxygen-containing mono- and sesquiterpenes (mainly 1,8-cineole) dominated in LN (50%), LA-SMA (88%) and LA-SMI (57%) and mono- and sesquiterpene hydrocarbons (mainly α-pinene) dominated in LA-PIC (61%) and LA-SJO (44%). Linalool and α-terpinyl acetate were also abundant in LA-SMA and trans-cinnamyl acetate in LA-SJO. These three components and all of the Laurus' EO showed high repulsive activity against medfly oviposition. In contact assays on medfly adults, a moderate degree of mortality was observed, being the most toxic samples, in decreasing order, trans-cinnamyl acetate > α-terpinyl acetate > LA-SMI ∼ LA-SMA > LA-SJO ∼ linalool. In fumigant assays, α-terpinyl acetate was the only compound that showed some toxicity on medfly adults. According to the obtained results, the Laurus EO seem promising to be used against medfly oviposition in integrated pest management strategy.     

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.     

Foliage sesquiterpenes and diterpenes of Podocarpus spicatus

Investigation of the foliage oils of 49 specimens of P. spicatus has shown marked infraspecific variation. Seven sesquiterpene hydrocarbons (δ-elemene, α-copaene, longifolene, germacrene-D, bicyclogermacrene, δ-cadinene and selina-4(14),7(11)-diene), two oxygenated sesquiterpenes (curzerenone and atractylon), ten diterpene hydrocarbons (rimuene, beyerene, sclarene, rosadiene, pimaradiene, sandaracopimaradiene, isopimara-7,15-diene, isopimara-8,15-diene, phyllocladene and kaurene), and two oxygenated diterpenes (8β-hydroxyisopimarene and ferruginol) have been isolated and identified. Of particular interest is the isolation of (+)-germacrene-D and (−)-δ-cadinene, along with the rarely reported curzerenone, atractylon, and (+)-rosadiene. This is the first report of selina-4(14),7(11)-diene in an optically active form. The occurrence of both enantiomers of sandaracopimaradiene is also of interest. The biosynthetic significance of correlations in the levels of the terpenes present is discussed.     

Essential oil of Bassia muricata: Chemical characterization,antioxidant activity,and allelopathic effect on the weed Chenopodium murale

The essential oil (EO) of Bassia muricata shoots was extracted via hydro-distillation and then investigated by gas chromatography-mass spectrometry. Thirty-four compounds were recognized for the first time from this plant, representing 100% of the total mass. Terpenoids represented the major components with 69.17% of the total mass, containing oxygenated sesquiterpenes (53.18%), oxygenated monoterpenes (9.77%), sesquiterpene hydrocarbons (5.03%), and diterpenes (1.19%). Additionaly, 6-methoxy-1-acetonaphthone was the only aromatic compound represented in a high percentage of the total identified compounds with 22.35%. Additionally, a percent of 8.48% of the total mass was hydrocarbons. Only one oxygenated sesquiterpene namely hexahydrofarnesyl acetone representing 47.35% of the total mass was identified. It was followed by methoxy-1-acetonaphthone (19.92%), n-dotriacontane (3.58%), endo-borneol (3.24%), 6-methy-α-ionone (3.04%), and α-gurjunene (2.65%). The EO exhibited moderate antioxidant activity comparable with ascorbic acid as a standard, where it attained IC₅₀ value of 20.70 µL L⁻¹ and 16.32 µL L⁻¹, for DPPH and ABTS. The EO of B. muricata significantly reduces the germination and seedling development of the weed Chenopodium murale. The EO showed an IC₅₀ value of 175.60 µL L⁻¹, 246.65 µL L⁻¹, and 308.33 µL L⁻¹ for root growth, shoot growth, and germination, respectively. Therefore, this EO could be a good green resource for the control of weeds.     

Casearia sylvestris Essential Oil Degradation Products Generated by Leaf Processing

Casearia sylvestris is an endemic tree of the Latin America that the essential oil (EO) has anti-inflammatory and gastroprotective actions. This study evaluates the chemical composition of the EO from the volatile fractions of in natura, fresh, and dried C. sylvestris var. sylvestris and var. lingua leaves. For both varieties, the dried leaves presented higher EO yield as compared to fresh leaves. The major EO chemical components were (E)-caryophyllene, α-humulene, germacrene D, bicyclogermacrene, spathulenol, caryophyllene oxide, and humulene epoxide II. In both varieties, the content of sesquiterpene hydrocarbons decreased and oxygenated sesquiterpenes increased on going from in natura to fresh and dried leaves, which indicated that leaf drying and hydrodistillation modified the volatile composition. The results also suggested that bicyclogermacrene and (E)-caryophyllene were oxidized during processing, to generate spathulenol and caryophyllene oxide, respectively. C. sylvestris varieties and in natura, fresh, and dried leaves differed in terms of the chemical composition of volatiles, which could affect the EO biological activities.     

Analysis of differentially expressed genes in response to endogenous cytokinins during cotton leaf senescence

Cytokinins have been implicated in delaying leaf senescence. We previously generated transgenic cotton (Gossypium hirsutum L.) plants that harbor the Agrobacterium isopentenyl transferase gene (ipt) directed by a proteinase gene promoter. Here, we report that mRNAs were isolated from ipt cotton leaves and azygous leaves and were subsequently sequenced using Illumina Solexa technology. The sequence tags were searched against the TIGR database and the related gene expression profiles were compared resulting in the identification of 1 218 differentially expressed genes (DEGs): 719 up-regulated and 499 down-regulated. Analyzing the DEGs in the ipt cotton leaves showed that these genes belonged to four pathways: flavone biosynthesis, arginine and proline metabolism, glyoxylate and dicarboxylate metabolism, and RNA degradation. These pathways increased the activities of antioxidants, inhibited the effect of ethylene, and prevented degradation of macromolecules during senescence. The expression patterns of 17 genes were evaluated by real-time PCR and results were in agreement with the patterns of sequencing analysis. The identification of the DEGs may help us to understand a role of cytokinins in leaf senescence.     

RNA-seq analysis reveals a key role of brassinolide-regulated pathways in NaCl-stressed cotton

Brassinolide (BL) alleviates salt injury in cotton seedlings; however, little is known about the molecular mechanisms of this response. In this study, digital gene expression analysis was performed to better understand the regulatory pathways of BL in NaCl-stressed cotton (Gossypium hirsutum L.). Compared with control plants (CK), a total of 1 162 and 7 659 differentially expressed genes (DEGs) were detected in the leaves and roots of NaCl-treated plants, respectively. Most of the DEGs in NaCl-treated plants, compared to CK, were regulated by BL. Moreover, expression patterns of DEGs in BL+NaCl treated plants were similar to those in CK plants; however, the responses of DEGs in the leaves and roots of NaCl-treated plants to BL differed. In the roots, BL-regulated DEGs were involved in protein biosynthesis, whereas in the leaves, BL promoted photosynthesis in NaCl-stressed cotton. BL treatment also significantly increased the overall biomass, chlorophyll a + b content in leaves, and the protein content in roots in NaCl-stressed cotton. The downregulation of stress-responsive genes in BL+NaCl-stressed leaves was also found. These results suggest that BL can alleviate NaCl injury in cotton plants.     

Chemical and phylogenetic relationships among Aristolochia L. (Aristolochiaceae) from southeastern Brazil

In this report, a molecular phylogeny based on the plastid gene matK and on the region between the genes trnL and trnF, and a chemical relationship based on the pattern of sesquiterpenes, is proposed for Aristolochia species from southeastern Brazil. We found that Aristolochia is a monophyletic genus and species considered to be derived contain labdanoic acids (LAs) in their leaves. The phenetic relationship recovered with sesquiterpenes did not agree with the phylogenetic relationships for Aristolochia, and three main clusters were recognized, namely, germacrene-D, germacrene-C and Z-caryophyllene groups. The presence of different sesquiterpene structures in species that are phylogenetically closely related may reflect adaptations to avoid predation of herbivores specialized in feeding on Aristolochiaceae plants.     

Interconversions of aglycone and host-selective toxin from Helminthosporium sacchari

Helminthosporium sacchari, a fungus that causes disease in sugarcane, produces oligosaccharide-sesquiterpene toxins (HS toxins A, B, and C) that are required for infection and disease development. Two free sesquiterpenes were isolated from mycelium but not from culture fluids of the fungus. One sesquiterpene was identified by HPLC and mass spectrometry as an aglycone of HS toxin C and could be obtained by enzymatic hydrolysis of this toxin. The other sesquiterpene appeared to be the 2-keto form of the first compound. The aglycone from toxin C hydrolysis was labelled with tritium by successive treatments with active manganese dioxide, sodium boro[³H]hydride, and lithium aluminium hydride. The labelled compound was fed to cultures of H. sacchari, radioactivity was incorporated into HS toxin C and into lower molecular weight homologues. The results suggest a metabolic route (aglycone → metabolite Y, → HS toxin → metabolite X) for the biosynthesis of HS toxin; metabolites X and Y are lower molecular weight homologues of the toxin.     

Sesquiterpenes from the conifer root rot pathogen Heterobasidion occidentale

Investigation of the production of secondary metabolites of Heterobasidion occidentale led to the isolation and identification of six sesquiterpenes (illudolone A and B, illudolactone A and B, deoxyfomannosin A and B) along with the well-known sesquiterpene fomannosin and the previously described benzohydrofuran fomannoxin. The structures and relative configurations of the compounds were determined by 1D and 2D NMR spectroscopic analysis as well as by HRMS. Their absolute configuration and biosynthesis were suggested and discussed in relation to fomannosin. Four compounds showed growth inhibiting activity against several basidiomycetes, Phlebiopsis gigantea, Phanerochaete chrysosporium and H. occidentale, and toxicity towards the moss Physcomitrella patens. In addition, one compound displayed activity against the bacterium Variovorax paradoxus as well as against the ascomycete Fusarium oxysporum.     

Biosynthesis of mono- and sesquiterpenes in peppermint from mevalonate-2-C

The volatile oil from peppermint (Mentha piperita L.) is composed primarily of monoterpenes with less than 2% sesquiterpenes. However, radioactivity from mevalonate-2-¹⁴C is incorporated into caryophyllene and other sesquiterpene hydrocarbons much more extensively than into monoterpenes by peppermint cuttings. Both mono- and sesquiterpenes show maximum incorporation of label after 6 hr (0·03% vs. 0·33% of the physiological isomer) and lose 75% of the incorporated label after an additional 6 hr. Caryophyllene derived from mevalonate-2-¹⁴C after 6 hr of incorporation was chemically degraded. The isoprenoid origin of caryophyllene was confirmed, and preferential labelling of the isopentenyl pyrophosphate derived portions of the molecule was noted. On the basis of such evidence it appears that separate sites may exist for the biosynthesis of mono- and sesquiterpenes and that an endogenous dimethylallyl pyrophosphate pool may participate in the biosynthesis of sesquiterpenes in peppermint.