Germacrene C synthase from Lycopersicon esculentum cv. VFNT Cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase (elemeneyterpenoid cyclaseysesquiterpene synthaseyfarnesyl diphosphate cyclization)

Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, b-caryophyllene, a-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases. With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp fragment that was used as a hybridization probe to screen the cDNA library. Thirty-one clones were evaluated for functional expression of terpenoid cyclase activity in Escherichia coli by using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA isolates expressed sesquiterpene synthase activity, and GC-MS analysis of the products identified germacrene C with smaller amounts of germacrene A, B, and D. None of the expressed proteins was active with geranylgeranyl diphosphate; however, one truncated protein converted geranyl diphosphate to the monoterpene limonene. The cDNA inserts specify a deduced polypeptide of 548 amino acids (Mr 5 64,114), and sequence comparison with other plant sesquiterpene cyclases indicates that germacrene C synthase most closely resembles cotton d-cadinene synthase (50% identity). Volatile metabolites of Lycopersicon species are of interest because of their roles in tomato flavor and in host defense against arthropod herbivores (1–5). Whereas few volatile terpenoids are found in fruit (1, 2), the leaf glandular trichomes produce a rich spectrum of monoterpenes and sesquiterpenes. Nearly 20 monoterpenes, including limonene, have been found in the leaves of the domestic tomato Lycopersicon esculentum (6, 7), and most are also present in wild tomato species (7). A number of C13 norsesquiterpenoid glycosidic ethers have been reported in tomato fruit (8); these compounds are derived by degradation of carotenoids (9). The sesquiterpene content of tomato leaf oil varies considerably among species, with caryophyllene and humulene being widespread and reported from L. esculentum, Lycopersicon hirsutum, Lycopersicon pimpinellifolium, Lycopersicon peruvianum, Lycopersicon cheesmanii, Lycopersicon chilense, and Lycopersicon chumielewski (7). The epoxides of both of these sesquiterpenes are also present in L. esculentum, as is a low level of d-elemene (6). Various accessions of L. hirsutum contain a-copaene, g-elemene, zingiberene, and a-santalene as major leaf oil sesquiterpenes (7). Germacrenes have not been reported in the genus Lycopersicon. The terpenoid composition of the highly disease-resistant L. esculentum cv. VFNT Cherry (a tomato of multispecies pedigree carrying resistance to Verticillium dahliae, Fusarium oxysporum, root-knot nematode, tobacco mosaic virus, and Alternaria stem canker) has not been examined, although it is an important breeding line (10). VFNT Cherry is a good model system for understanding the molecular basis of sesquiterpene biosynthesis and of glandular trichome-based expression of defense genes from which novel forms of resistance could be developed. In this report, we describe the monoterpene and sesquiterpene composition of VFNT Cherry tomato leaves, leaf sesquiterpene cyclase enzymology, the PCR strategy used to isolate a cDNA encoding germacrene C synthase, and the comparison of this sesquiterpene cyclase to other plant terpenoid cyclases. MATERIALS AND METHODS Experimental Materials. L. esculentum cv. VFNT Cherry tomato plants (obtained as seed from John Steffens, Cornell University) were propagated from cuttings and grown under conditions as reported (11). L. esculentum cv. Better Boy plants were obtained from C.A. Ryan (Washington State University) and similarly propagated. Methods have been reported for the preparation of [1-3H]geranyl diphosphate (GDP; 122 Ciymol; 1 Ci 5 37 GBq) (12), [1-3H]farnesyl diphosphate (FDP; 125 Ciymol) (13), and [1-3H]geranylgeranyl diphosphate (GGDP; 90 Ciymol) (14). Terpenoid standards were from our own collection or gifts from Bob Adams (Baylor University) or Larry Cool (University of California, Berkeley). The VFNT Cherry tomato cDNA library, derived from epidermisenriched young leaf tissue, was also a gift from John Steffens. All other biochemicals were purchased from Sigma or Aldrich, unless otherwise noted. Volatile Terpene Analysis. Expanding tomato leaves were harvested, frozen in liquid N2, and then subjected to simultaneous steam distillation and solvent (pentane) extraction (15) using the J & W Scientific apparatus. The resulting pentane phase, collected at 0–4°C, was passed over a column of The 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. © 1998 by The National Academy of Sciences 0027-8424y98y952216-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: FDP, farnesyl diphosphate; GDP, geranyl diphosphate; GGDP, geranylgeranyl diphosphate; TEAS, tobacco 5-epi-aristolochene synthase. Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AF035630 and AF035631). †Current address: Hitachi Software, 601 Gateway Boulevard, Suite 500, South San Francisco, CA 94080. §To whom reprint requests should be addressed. e-mail: croteau@ mail.wsu.edu.