Rose scent: genomics approach to discovering novel floral fragrance-related genes.
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For centuries, rose has been the most important crop in the floriculture industry; its economic importance also lies in the use of its petals as a source of natural fragrances. Here, we used genomics approaches to identify novel scent-related genes, using rose flowers from tetraploid scented and nonscented cultivars. An annotated petal EST database of approximately 2100 unique genes from both cultivars was created, and DNA chips were prepared and used for expression analyses of selected clones. Detailed chemical analysis of volatile composition in the two cultivars, together with the identification of secondary metabolism-related genes whose expression coincides with scent production, led to the discovery of several novel flower scent-related candidate genes. The function of some of these genes, including a germacrene D synthase, was biochemically determined using an Escherichia coli expression system. This work demonstrates the advantages of using the high-throughput approaches of genomics to detail traits of interest expressed in a cultivar-specific manner in nonmodel plants. EST sequences were submitted to the GenBank database (accession numbers BQ 103855 to BQ 106728). (+info)
Terpenoid metabolism in wild-type and transgenic Arabidopsis plants.
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Volatile components, such as terpenoids, are emitted from aerial parts of plants and play a major role in the interaction between plants and their environment. Analysis of the composition and emission pattern of volatiles in the model plant Arabidopsis showed that a range of volatile components are released, primarily from flowers. Most of the volatiles detected were monoterpenes and sesquiterpenes, which in contrast to other volatiles showed a diurnal emission pattern. The active terpenoid metabolism in wild-type Arabidopsis provoked us to conduct an additional set of experiments in which transgenic Arabidopsis overexpressing two different terpene synthases were generated. Leaves of transgenic plants constitutively expressing a dual linalool/nerolidol synthase in the plastids (FaNES1) produced linalool and its glycosylated and hydroxylated derivatives. The sum of glycosylated components was in some of the transgenic lines up to 40- to 60-fold higher than the sum of the corresponding free alcohols. Surprisingly, we also detected the production and emission of nerolidol, albeit at a low level, suggesting that a small pool of its precursor farnesyl diphosphate is present in the plastids. Transgenic lines with strong transgene expression showed growth retardation, possibly as a result of the depletion of isoprenoid precursors in the plastids. In dual-choice assays with Myzus persicae, the FaNES1-expressing lines significantly repelled the aphids. Overexpression of a typical cytosolic sesquiterpene synthase resulted in the production of only trace amounts of the expected sesquiterpene, suggesting tight control of the cytosolic pool of farnesyl diphosphate, the precursor for sesquiterpenoid biosynthesis. This study further demonstrates the value of Arabidopsis for studies of the biosynthesis and ecological role of terpenoids and provides new insights into their metabolism in wild-type and transgenic plants. (+info)
Nitrogen supply influences herbivore-induced direct and indirect defenses and transcriptional responses in Nicotiana attenuata.
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Although nitrogen (N) availability is known to alter constitutive resistance against herbivores, its influence on herbivore-induced responses, including signaling pathways, transcriptional signatures, and the subsequently elicited chemical defenses is poorly understood. We used the native tobacco, Nicotiana attenuata, which germinates in the postfire environment and copes with large changes in soil N during postfire succession, to compare a suite of Manduca sexta- and elicitor-induced responses in plants grown under high- and low-N (LN) supply rates. LN supply decreased relative growth rates and biomass by 35% at 40 d compared to high-N plants; furthermore, it also attenuated (by 39 and 60%) the elicitor-induced jasmonate and salicylate bursts, two N-intensive direct defenses (nicotine and trypsin proteinase inhibitors, albeit by different mechanisms), and carbon-containing nonvolatile defenses (rutin, chlorogenic acid, and diterpene glycosides), but did not affect the induced release of volatiles (cis-alpha-bergamotene and germacrene A), which function as indirect defenses. M. sexta and methyl jasmonate-induced transcriptional responses measured with a microarray enriched in herbivore-induced genes were also substantially reduced in plants grown under LN supply rates. In M. sexta-attacked LN plants, only 36 (45%) up-regulated and 46 (58%) down-regulated genes showed the same regulation as those in attacked high-N plants. However, transcriptional responses frequently directly countered the observed metabolic changes. Changes in a leaf's sensitivity to elicitation, an attacked leaf's waning ability to export oxylipin wound signals, and/or resource limitations in LN plants can account for the observed results, underscoring the conclusion that defense activation is a resource-intensive response. (+info)
Content and composition of the essential oil of Thymus serpyllum L. growing wild in Estonia.
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OBJECTIVE: The aim of this work was to analyze the essential oil content and its composition in the drug (Serpylli herba) of wild thyme (Thymus serpyllum L.) originating from 20 different natural places of growth in Estonia. MATERIAL AND METHODS: The quantitative content of essential oil was determined according to the method of European Pharmacopoeia. Gas chromatographic analysis of essential oils was carried out using a gas chromatography with flame ionization detector on two fused silica capillary columns with bonded stationary phases NB-30 and SW-10. The identification of the oil components was accomplished by comparing their retention indices on two columns with the retention indices values of standard compounds, with our retention indices data bank and with literature data. The results obtained were confirmed by gas chromatography / mass spectrometry. RESULTS: The content of essential oil is between 0.6 and 4 ml/kg and usually is not in conformity with European Pharmacopoeia standard (3 ml/kg). There were 55 components identified in the essential oil of wild thyme of Estonian origin. Differently from the data in the literature of foreign countries, thymol and carvacrol are not the main components of the essential oil of wild thyme growing in Estonia. The main components here are (E)-nerolidol, caryophyllene oxide, myrcene, (E)-beta-caryophyllene and germacrene D. CONCLUSION: In Estonia, the (E)-nerolidol-caryophyllene oxide, (E)-nerolidol-myrcene and myrcene chemotypes of wild thyme drug are distinguishable. (+info)
A potent apoptosis-inducing activity of a sesquiterpene lactone, arucanolide, in HL60 cells: a crucial role of apoptosis-inducing factor.
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Six main sesquiterpene lactones (germacranolides) from Calea urticifolia were evaluated for in vitro cytotoxicity against human tumor cell lines HL60 and SW480 cells. Among them, arucanolide and parthenolide displayed marked cytotoxicity against both cell lines. Arucanolide exhibited a low IC(50) in HL60 cells. The cytotoxic activity of arucanolide was observed at lower concentrations compared to that of parthenolide, which has been reported to be a typical and simple germacranolide. The activity was found to be mainly due to apoptosis that was assessed by morphological findings, DNA ladder formation (24 - 36 h), and flow cytometric analysis in HL60 cells. Western blotting and an apoptosis inhibition assay using caspase inhibitors did not demonstrate the activation of any caspases tested. However, the mitochondrial membrane potential of HL60 cells was lost after 24-h treatment with arucanolide, and concurrently apoptosis-inducing factor (AIF) released from mitochondria was detected by Western blot analysis. The inactivation of nuclear factor-kappaB, which has been commonly shown in parthenolide-induced apoptosis, did not occur in arucanolide-induced apoptosis. Taken together, the findings presented here indicate that arucanolide induced marked apoptosis in HL60 cells mainly by dissipating mitochondrial membrane potential, which would trigger AIF-induced apoptosis. (+info)
Inhibition of preadipocyte differentiation by germacranolides from Calea urticifolia in 3T3-L1 cells.
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The effects of germacranolides isolated from Calea urticifolia on adipocytic differentiation of 3T3-L1 cells were examined. These germacranolides inhibited adipogenesis at a concentration of 1.25-5 microM. But no inhibitory activity against cell proliferation and no nonspecific binding activity to protein were observed. These results indicate that these germacranolides are the specific inhibitors of preadipocyte differentiation. (+info)
Cytotoxic germacranolide sesquiterpene from Inula cappa.
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A new germacranolide, inulacappolide (1), was isolated from the EtOH extract of the whole plant of Inula cappa along with 16 known compounds. The structure of inulacappolide was a rare 1(10)-saturated type of germacran-6,12-olide, identified as 2alpha-acetoxy-3beta-hydroxy-9beta-angeloyloxygermacra-4-en-6alpha,12-olide by spectral analysis (IR, HR-ESI/MS, (1)H-NMR, (13)C-NMR, HMQC, HMBC, NOESY). In vitro, it showed antiproliferative effects against human cervical cancer HeLa, human leukemia K562 and human nasopharyngeal carcinoma KB cell lines with IC(50) values of 1.2 microM, 3.8 microM and 5.3 microM, respectively. (+info)
Interception of the enzymatic conversion of farnesyl diphosphate to 5-epi-aristolochene by using a fluoro substrate analogue: 1-fluorogermacrene A from (2E,6Z)-6-fluorofarnesyl diphosphate.
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Tobacco 5-epi-aristolochene synthase (TEAS) catalyzes the Mg(II)-dependent cyclizations and rearrangements of (E,E)-farnesyl diphosphate (PP) to the bicyclic sesquiterpene hydrocarbon via a tightly bound (+)-germacrene A as a deprotonated intermediate. With the native enzyme, only a few percent of the putative germacrene A intermediate is released from the active site during the catalytic cycle. 6-Fluorofarnesyl PP was designed and synthesized with the aim of arresting the cyclization-rearrangement mechanism en route to 5-epi-aristolochene. Indeed, incubation of (2E,6Z)-6-fluorofarnesyl PP with recombinant TEAS afforded (-)-1-fluorogermacrene A as the sole product in 58% yield. Steady-state kinetic experiments with farnesyl PP and the 6-fluoro analogue showed that the overall catalytic efficiencies (k(cat)/K(m)) are essentially the same for both substrates. 1-Fluorogermacrene A was characterized by chromatographic properties (TLC, GC), MS, optical rotation, UV, IR and (1)H NMR data, and by heat-induced Cope rearrangement to (+)-1-fluoro-beta-elemene. (1)H NMR spectra at room temperature revealed that this (E,E)-configured fluorocyclodecadiene exists in solution as a 7:3 mixture of UU and UD conformers. 1-Fluorogermacrene A underwent trifluoroacetic acid-catalyzed cyclization to give three 1alpha-fluoroselinene isomers at a rate estimated to be about 1000 times slower than that of the similar cyclization of (+)-germacrene A to the parent selinenes. (+info)