Terpenoid-based defenses in conifers: cDNA cloning, characterization, and functional expression of wound-inducible (E)-a-bisabolene synthase from grand fir (Abies grandis) [sesquiterpene synthaseyjuvenile hormone analogueyplant defense geney(E)-4-(1,5-dimethyl-1,4-hexadienyl)-1-methylcyclohexeneytodomatuic acid]

E)-a-Bisabolene synthase is one of two wound-inducible sesquiterpene synthases of grand fir (Abies grandis), and the olefin product of this cyclization reaction is considered to be the precursor in Abies species of todomatuic acid, juvabione, and related insect juvenile hormone mimics. A cDNA encoding (E)-a-bisabolene synthase was isolated from a wound-induced grand fir stem library by a PCR-based strategy and was functionally expressed in Escherichia coli and shown to produce (E)-a-bisabolene as the sole product from farnesyl diphosphate. The expressed synthase has a deduced size of 93.8 kDa and a pI of 5.03, exhibits other properties typical of sesquiterpene synthases, and resembles in sequence other terpenoid synthases with the exception of a large aminoterminal insertion corresponding to Pro81–Val296. Biosynthetically prepared (E)-a-[3H]bisabolene was converted to todomatuic acid in induced grand fir cells, and the time course of appearance of bisabolene synthase mRNA was shown by Northern hybridization to lag behind that of mRNAs responsible for production of induced oleoresin monoterpenes. These results suggest that induced (E)-a-bisabolene biosynthesis constitutes part of a defense response targeted to insect herbivores, and possibly fungal pathogens, that is distinct from induced oleoresin monoterpene production. The oleoresin of conifers is a complex mixture of volatile monoterpenes and sesquiterpenes (turpentine) and nonvolatile diterpene resin acids (rosin) that has arisen during 300 million years of conifer evolution (1–3) as a multifunctional defense against insect herbivores and fungal pathogens. The turpentine fraction of conifer oleoresin may contain up to 30 different monoterpenes (4) and an even larger number of sesquiterpenes (5). These volatiles are toxic to bark beetles (Scolytidae) and their pathogenic fungal symbionts and also furnish the solvent for diterpene resin acids that harden to form a mechanical barrier to seal wound sites upon evaporation of resin volatiles (6). In grand fir (Abies grandis), increased formation of oleoresin monoterpenes, sesquiterpenes and diterpenes is induced by insect attack (7–10), and this inducible defense response is mimicked by mechanically wounding sapling stems (8, 9, 11). Therefore, grand fir has been developed as a model system for the study of the regulation of defensive terpene biosynthesis in conifers (12–15). The induced production of oleoresin monoterpenes and diterpene resin acids has been well defined (11, 16–18). Recent cDNA cloning of abietadiene synthase (19), the major wound-inducible diterpene synthase of grand fir, and of three wound-inducible monoterpene synthases (13), led to the structural analysis of the corresponding catalysts and to the molecular dissection of induced monoterpene and diterpene biosynthesis (14, 15). Comparatively little is known about the kinetics and functional significance of sesquiterpene biosynthesis in conifers. Sesquiterpenes of the juvabione family, based on the bisabolane skeleton (Fig. 1), mimic insect juvenile hormones and, thus, can disrupt insect development and reproduction (20, 21). Juvabione [the methyl ester of todomatuic acid (Fig. 1)] is a component of ‘‘paper factor,’’ a mixture of oxygenated bisabolane sesquiterpenes that is responsible for the failure of certain insects to molt into adults when reared in contact with paper made from fir species (genus Abies) (22–25). (E)-aBisabolene synthase is one of the two inducible sesquiterpene synthases of grand fir that, after stem wounding, is responsible for a dramatic shift in product composition as determined by in vitro assay (5). Induced sesquiterpenes could form a second line of chemical defense against insect herbivores and fungal pathogens that is distinct from the initial defense provided by inducible monoterpene production. Todomatuic acid (Fig. 1) has been reported in grand fir, accumulates in response to aphid feeding (10), and has both juvenile hormone activity against Tenebrio larvae (26) and antifungal activity (27, 28). Given the obvious structural relationship between bisabolene and todomatuic acid (Fig. 1), these observations, together with the reported wound induction of bisabolene synthase (5), suggest that de novo biosynthesis of todomatuic acid may represent an inducible defense against insect herbivores and fungal pathogens. In this paper, we describe the cDNA cloning and characterization of grand fir ag1, a member of the Tpsd subfamily of gymnosperm terpene synthases (13, 14), and we report on the bacterial expression of the encoded sesquiterpene synthase, which yields (E)-a-bisabolene as the sole product from farnesyl diphosphate. Northern hybridization experiments revealed a wound-induced, transient increase of bisabolene synthase mRNA with a time course of accumulation different from that of the transcriptional activation of monoterpene synthase genes. MATERIALS AND METHODS Substrates and Reagents. [1-3H]Geranyl diphosphate (GDP; 250 Ciymol; 1 Ci 5 37 GBq) (29), [1-3H]farnesyl diphosphate 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 0027-8424y98y956756-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: FDP, farnesyl diphosphate; GDP, geranyl diphosphate; GGDP, geranylgeranyl diphosphate; RACE, rapid amplification of cDNA ends. Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AF006194 and AF006195). *Present address: Max-Planck-Institut für Chemische Ökologie, D-07745 Jena, Germany. †J.B. and J.C. contributed equally to this work. ‡Present address: Universität Würzburg, Lehrstuhl für Botanik II, D-97082 Würzburg, Germany. §To whom reprint requests should be addressed. e-mail: croteau@ mail.wsu.edu.