Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-b-farnesene (insect pheromoneyplant-insect chemical communicationysynomone)

E)-b-Farnesene is a sesquiterpene semiochemical that is used extensively by both plants and insects for communication. This acyclic olefin is found in the essential oil of peppermint (Mentha x piperita) and can be synthesized from farnesyl diphosphate by a cell-free extract of peppermint secretory gland cells. A cDNA from peppermint encoding (E)-b-farnesene synthase was cloned by random sequencing of an oil gland library and was expressed in Escherichia coli. The corresponding synthase has a deduced size of 63.8 kDa and requires a divalent cation for catalysis (Km for Mg21 ' 150 mM; Km for Mn21 ' 7 mM). The sesquiterpenoids produced by the recombinant enzyme, as determined by radio-GC and GC-MS, are (E)-b-farnesene (85%), (Z)-b-farnesene (8%), and dcadinene (5%) with the native C15 substrate farnesyl diphosphate (Km ' 0.6 mM; Vrel 5 100) and Mg21 as cofactor, and (E)-b-farnesene (98%) and (Z)-b-farnesene (2%) with Mn21 as cofactor (Vrel 5 80). With the C10 analog, GDP, as substrate (Km 5 1.5 mM; Vrel 5 3 with Mg21 as cofactor), the monoterpenes limonene (48%), terpinolene (15%), and myrcene (15%) are produced. (E)-b-farnesene (Fig. 1) is an acyclic sesquiterpene olefin that occurs in a wide range of both plant and animal taxa. More than 600 papers have been published on the occurrence of this natural product and its deployment as an important courier in chemical communication. The olefin is found in the essential oil of hundreds of species of both gymnosperms, such as Torreya taxifolia (Florida torreya) (1) and Larix leptolepis (larch) (2), and angiosperms, such as Robinia pseudoacacia (black locust) (3), Medicago sativa (alfalfa) (4), Chamomilla recutita (chamomile) (5), Vitis vinifera (grapes) (6), Cannabis sativa (hemp) (7), Zea mays (corn) (8), Piper nigrum (black pepper)†, Daucus carota (carrot)†, and Mentha x piperita (peppermint) (9). Although socially dominant male mice produce both afarnesene and (E)-b-farnesene in their urine as pheromones (10), it is in the insects and plants that the use of (E)-bfarnesene as a semiochemical is most extensive. (E)-bFarnesene is emitted by the Dufour’s gland of andrenid bees (11) and by several genera of ants (12–14), where it serves both as a defensive allomone and as a trail pheromone. This sesquiterpene is synthesized de novo in the osmetrial glands of larval Papilio (LepidopterayPapilionidae) as an allomone (15), and it functions as a feeding stimulant to the sand fly Lutzomyia longipalpis (DipterayPsychodidae), an important vector of the blood disease leishmaniasis (16). Several species of predatory carabid beetles use (E)-b-farnesene as a prey-finding kairomone (17). When released by corn, this olefin is also a kairomonal oviposition stimulant to the European corn borer (Ostrinia) (18). (E)-b-farnesene is the major component of pollen odor in Lupinus and stimulates pollination behavior in bumblebees (19). Feeding by larval lepidopterans, such as Heliothis or Spodoptera (Noctuidae), increases the amount of (E)-b-farnesene released by corn; the volatile olefin then is detected as a synomone by the parasitic wasp Cotesia marginiventris (HymenopterayBraconidae) for locating the lepidopteran hosts (8). Circumstantial evidence also suggests the lepidopteran induced production and emission of (E)-bfarnesene from corn serves as a synomone for Cotesia kariyai (20) and from cotton leaves as a synomone for C. marginiventris (21, 22). Perhaps of greatest significance in plant-insect interactions is the use of (E)-b-farnesene by most aphid species as an alarm pheromone (23, 24). Aphids exposed to (E)-b-farnesene become agitated and disperse from their host plant (25). Alate aphids are usually more sensitive than are apterae species and often will not colonize a host displaying (E)-b-farnesene. Ants that defend aphids are sensitive to host-emitted (E)-bfarnesene and, when exposed, will display aggressive behavior (26). (E)-b-farnesene also mimics the action of juvenile hormone III in some insects (27), may play a role in control of aphid morphological types, and is acutely toxic to aphids at a dose of 100 ngyaphid (28). (E)-b-farnesene vapor is also toxic to whiteflies (29). Efforts to control aphid behavior by topical application of (E)-b-farnesene to crops have met with little success, because of volatility and rapid oxidative inactivation in air (30). Derivatives of (E)-b-farnesene with reduced volatility, or increased stability, have shown promise in reducing aphidtransmitted viruses, such as barley mosaic virus (30), potato virus Y (31), and beet mosaic virus (31). The wild potato Solanum berthaultii, which produces (E)-b-farnesene in type A trichomes, is more repellent to the green peach aphid than are commercial varieties of S. tuberosum that produce lower levels of the olefin (32, 33). In alfalfa, repellency to the blue alfalfa aphid and the pea aphid is correlated with the leaf content of (E)-b-farnesene, but not with the amount of the co-occurring sesquiterpene caryophyllene (34). For plants that produce (E)-b-farnesene, breeding for increased production has met with some success (34), but has been limited by genetic variation in these species. (E)-bfarnesene synthase has been purified from maritime pine (Pinus pinaster) and characterized (35), but the gene has not yet been isolated from any source. A cDNA clone for (E)-bThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y9412833-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: FDP, farnesyl diphosphate; I, identity; S, similarity. Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF024615). *To whom reprint requests should be addressed. e-mail: croteau@ mail.wsu.edu. †Beckstrom-Sternberg, S. M. & Duke, J. A. (1994) The Phytochemical Database, Version 4.3; http:yyprobe.nalusda.gov:8300ycgi-biny browseyphytochemdb.