The plant sesquiterpene germacrene D specifically activates a major type of antennal receptor neuron of the tobacco budworm moth Heliothis virescens.
Plants release hundreds of volatiles that are important in interactions with insects or other organisms. However, knowledge is scarce as to which of the compounds are detected by the organism's olfactory receptor neurons. In the present study, single receptor neurons on the antennae of the tobacco budworm moth, Heliothis virescens, were screened for their sensitivities to naturally produced plant volatiles by the use of gas chromatography linked to electrophysiological recordings from single cells (GC-SCR). Plant volatiles, collected by aeration of host and non-host plants, were tested on each receptor neuron via parallel GC-columns. Thus, simultaneous recordings of the gas chromatogram and the neuron responses to each component were obtained. One type of receptor neuron, appearing in 80% of all experiments, responded with high sensitivity and selectivity to one particular component, present in host as well as non-host mixtures. The component, identified as a sesquiterpene hydrocarbon by linked gas chromatography-mass spectrometry, was isolated from a sesquiterpene fraction of cubebe oil and identified by NMR as germacrene D. The purified compound was then re-tested via gas chromatography on the same receptor neuron type, verifying the identification. A weaker response to another sesquiterpene hydrocarbon was also recorded. (+info)
Molecular characterization of tomato 3-dehydroquinate dehydratase-shikimate:NADP oxidoreductase.
Analysis of cDNAs encoding the bifunctional 3-dehydroquinate dehydratase-shikimate:NADP oxidoreductase (DHQase-SORase) from tomato (Lycopersicon esculentum) revealed two classes of cDNAs that differed by 57 bp within the coding regions, but were otherwise identical. Comparison of these cDNA sequences with the sequence of the corresponding single gene unequivocally proved that the primary transcript is differentially spliced, potentially giving rise to two polypeptides that differ by 19 amino acids. Quantitative real-time polymerase chain reaction revealed that the longer transcript constitutes at most 1% to 2% of DHQase-SORase transcripts. Expression of the respective polypeptides in Escherichia coli mutants lacking the DHQase or the SORase activity gave functional complementation only in case of the shorter polypeptide, indicating that skipping of a potential exon is a prerequisite for the production of an enzymatically active protein. The deduced amino acid sequence revealed that the DHQase-SORase is most likely synthesized as a precursor with a very short (13-amino acid) plastid-specific transit peptide. Like other genes encoding enzymes of the prechorismate pathway in tomato, this gene is elicitor-inducible. Tissue-specific expression resembles the patterns obtained for 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase 2 and dehydroquinate synthase genes. This work completes our studies of the prechorismate pathway in that cDNAs for all seven enzymes (including isozymes) of the prechorismate pathway from tomato have now been characterized. (+info)
Biosynthesis of germacrene A carboxylic acid in chicory roots. Demonstration of a cytochrome P450 (+)-germacrene a hydroxylase and NADP+-dependent sesquiterpenoid dehydrogenase(s) involved in sesquiterpene lactone biosynthesis.
Sprouts of chicory (Cichorium intybus), a vegetable grown in the dark, have a slightly bitter taste associated with the presence of guaianolides, eudesmanolides, and germacranolides. The committed step in the biosynthesis of these compounds is catalyzed by a (+)-germacrene A synthase. Formation of the lactone ring is the postulated next step in biosynthesis of the germacrene-derived sesquiterpene lactones. The present study confirms this hypothesis by isolation of enzyme activities from chicory roots that introduce a carboxylic acid function in the germacrene A isopropenyl side chain, which is necessary for lactone ring formation. (+)-germacrene A is hydroxylated to germacra-1(10),4,11(13)-trien-12-ol by a cytochrome P450 enzyme, and is subsequently oxidized to germacra-1(10),4,11(13)-trien-12-oic acid by NADP+-dependent dehydrogenase(s). Both oxidized germacrenes were detected as their Cope-rearrangement products elema-1,3,11(13)-trien-12-ol and elema-1,3,11(13)-trien-12-oic acid, respectively. The cyclization products of germacra-1(10),4,11(13)-trien-12-ol, i.e. costol, were also observed. The (+)-germacrene A hydroxylase is inhibited by carbon monoxide (blue-light reversible), has an optimum pH at 8.0, and hydroxylates beta-elemene with a modest degree of enantioselectivity. (+info)
Receptor neuron discrimination of the germacrene D enantiomers in the moth Helicoverpa armigera.
Plants release complex mixtures of volatiles, including chiral constituents. In the search for the biologically relevant plant odorants, gas chromatography linked to electrophysiological recordings from single receptor neurons has been employed. In heliothine moths, including the females of the Eurasian cotton bollworm moth Helicoverpa armigera, a major type of receptor neurons is identified, showing high sensitivity and selectivity for the sesquiterpene germacrene D. In the present study, gas chromatography with a chiral column linked to single cell recordings were performed. It was found that all germacrene D neurons belonged to one type; all responded to both enantiomers, but (-)-germacrene D had approximately 10 times stronger effect than (+)-germacrene D. Parallel dose-response curves for the two enantiomers were obtained by direct stimulations. The enantiomeric composition of germacrene D, which differed in six plant species and in different individuals of one species, was determined on the basis of the neuron responses. The results, showing the presence of one neuron type for receiving the information about germacrene D in the various plants, suggests that the two enantiomers mediate the same kind of information to the moth, but with different intensity. (+info)
Biosynthesis of costunolide, dihydrocostunolide, and leucodin. Demonstration of cytochrome p450-catalyzed formation of the lactone ring present in sesquiterpene lactones of chicory.
Chicory (Cichorium intybus) is known to contain guaianolides, eudesmanolides, and germacranolides. These sesquiterpene lactones are postulated to originate from a common germacranolide, namely (+)-costunolide. Whereas a pathway for the formation of germacra-1(10),4,11(13)-trien-12-oic acid from farnesyl diphosphate had previously been established, we now report the isolation of an enzyme activity from chicory roots that converts the germacrene acid into (+)-costunolide. This (+)-costunolide synthase catalyzes the last step in the formation of the lactone ring present in sesquiterpene lactones and is dependent on NADPH and molecular oxygen. Incubation of the germacrene acid in the presence of 18O2 resulted in the incorporation of one atom of 18O into (+)-costunolide. The label was situated at the ring oxygen atom. Hence, formation of the lactone ring most likely occurs via C6-hydroxylation of the germacrene acid and subsequent attack of this hydroxyl group at the C12-atom of the carboxyl group. Blue light-reversible CO inhibition and experiments with cytochrome P450 inhibitors demonstrated that the (+)-costunolide synthase is a cytochrome P450 enzyme. In addition, enzymatic conversion of (+)-costunolide into 11(S),13-dihydrocostunolide and leucodin, a guaianolide, was detected. The first-mentioned reaction involves an enoate reductase, whereas the formation of leucodin from (+)-costunolide probably involves more than one enzyme, including a cytochrome P450 enzyme. (+info)
Glandular hairs of Sigesbeckia jorullensis Kunth (Asteraceae): morphology, histochemistry and composition of essential oil.
Long-stalked glandular hairs of outer and inner involucral bracts of Sigesbeckia jorullensis, which are important for epizoic fruit propagation, were investigated using light and scanning electron microscopy. The essential oil secreted by the hairs was analysed by chromatographic methods including gas chromatography/mass spectrometry and with a laser microprobe mass analyser. The glandular hairs consisted of a large multicellular stalk and a multicellular secreting head. The apical layer of glandular head cells was characterized by leucoplasts and calcium oxalate crystals. Below the apical cells there were up to six layers of cells containing many chloroplasts around the nucleus and surrounded by vacuoles filled with flavonoids and tannins. The essential oil originating in the head cells was secreted into the subcuticular space and may be liberated by rupture of the cuticle. It was mainly composed of sesqui- and diterpenes, with the sesquiterpene hydrocarbon germacrene-D as the main component. Monoterpenes, n-alkanes and their derivatives as well as flavonoid aglycones were also detected. The stickiness of the essential oil is probably associated with the high content of oxygenated sesqui- and diterpenes. In addition to long-stalked trichomes, small biseriate trichomes occurred, secreting small quantities of essential oil into a subcuticular space. (+info)
(-)-Germacrene D increases attraction and oviposition by the tobacco budworm moth Heliothis virescens.
The sesquiterpene germacrene D (GD) activates a major type of olfactory receptor neuron on the antennae of the heliothine moths. In Heliothis virescens females, 80% of the recordings have shown activity of one neuron type responding with high sensitivity and selectivity to GD. With the aim of determining the behavioural significance of this sesquiterpene, we have used a two-choice wind-tunnel to study the preference of mated H. virescens females for host plants with and without (-)-GD added. Tobacco plants containing dispensers with low release rate of (-)-GD had a greater attractiveness than tobacco plants without this substance. In addition, a significant increase of oviposition was found on the plants with (-)-GD. (+info)
Seven germacranolides, eupaglehnins A, B, C, D, E, and F, and 2alpha-acetoxyepitulipinolide from Eupatorium glehni.
Four new germacrane-type sesquiterpenoids with unsaturated acids as esters at the 8-position, two chlorine atom-containing lactones, 2alpha-acetoxyepitulipinolide, and 12 previously known compounds have been isolated from the MeOH extract of Eupatorium glehni (Compositae) and their structures have been determined on the basis of spectral data analyses. (+info)