Biosynthesis of the diterpenes labdenediol andsclareol from all trans-geranylgeranyl pyrophosphate was observed in cell-free extracts prepared from leaf midvein epidermal peels of Nicotiana glutinosa 24A. The bioactivities were shown to be exclusively localized in trichomes, to be conferred by a soluble enzyme(s), and to resemble other plant terpene cyclase activities in basic characteristics. Chromatographic methods for protein purification including gel filtration, anion exchange, hydroxyapatite chromatography, and free-flow isoelectric focusing. Thermal inactivation and end-product inhibition experiments did not afford separation of the biosynthetic activities. Results indicate that these labdane diterpenes are direct products of cyclization reactions catalysed by a soluble cyclase(s). To the best of our knowledge, this report represents the first case for direct biosynthesis of diterpene di-alcohols by cyclization of the acyclic precursor. A unified scheme for formation of labdane mono-and di-alcohols in Nicotiana species is presented.
Bioassays employingTetranychus urticae Koch were used to examine repellency of leaves and trichome secretions of one cultivar of tomato,Lycopersicon esculentum, and 11 accessions ofL. hirsutum consisting of seven accessions ofL. hirsutum f.typicum and four accessions ofL. hirsutum f.glabratum. Leaves of f.typicum were more repellent to mites than were those of f.glabratum orL. esculentum. Removal of trichomes and their secretions by wiping leaves of f.typicum accessions with 95% ethanol rendered them less repellent, indicating that trichomes or their secretions were potentially responsible for repellency. Gas and thin-layer chromatography of leaflet washes obtained by steeping leaflets in hexane indicated that the chemical composition of the washes differed among accessions. The main difference between botanical forms was the presence in leaflet washes from f.typicum accessions of compounds eluting late on the gas Chromatograph and migrating further on reverse-phase thin-layer chromatography; compounds having these characteristics were absent in washes of f.glabratum andL. esculentum. The compositions of secretions taken directly from type I, type IV, and type VI trichomes were similar to that of the leaflet wash obtained from the same f.typicum plant, indicating that leaflet washes contained mainly compounds of trichomal origin. Leaflet washes from f.typicum plants were generally more repellent toT. urticae than were washes from f.glabratum when tested by choice and nonchoice bioassays. Taken together, the data indicate that trichome secretions of f.typicum are likely responsible for potent repellency to two-spotted spider mites observed in this taxon; the likely active compounds are sesquiterpene acids or other polar constituents of trichome secretions.
Lycopersicon hirsutum, a wild relative of the tomato, is highly resistant to arthropod herbivores. Both botanic forms ofL. hirsutum, L. hirsutum f.glabratum (C.H. Mull.) andL. hirsutum f.typicum (Humb. & Bonpl.), are resistant to two-spotted spider mites,Tetranychus urticae Koch. However, leaves and trichome secretions from f.typicum repel mites more so than those from f.glabratum. We have previously demonstrated that trichome secretions from LA 1363 and LA 1927, accessions of f.typicum, repelled mites. In this paper we report the identification of the primary component of trichome secretions responsible for repellency. Leaflet washes having compositions similar to trichome secretions were collected and separated into neutral and acid fractions; repellency was mainly associated with the acid fraction, which, when applied to nonrepellent leaflets of f.glabratum, rendered them repellent. Separation of leaflet washes by HPLC allowed purification and subsequent identification by gas chromatography-mass spectrometry and nuclear magnetic resonance of 2,3-dihydrofamesoic acid (3,7,11-trimethyl-6, 10-dodecadienoic acid) as the primary chemical component responsible for repellency. Application of this acid to leaflets ofL. esculentum rendered them repellent. Other volatile compounds present in minor amounts in the acid fractions were farnesoic acid and 16∶0, 16∶3, 18∶0, 18∶2, and 18∶3 fatty acids. This is the first report of the natural occurrence of 2,3-dihydrofarnesoic acid.
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