Plant Molecular Biology 51: 119–133, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands. 119 Traumatic resin defense in Norway spruce (Picea abies): Methyl jasmonate-induced terpene synthase gene expression, and cDNA cloning and functional characterization of (+)-3-carene synthase Jenny Fäldt, Diane Martin, Barbara Miller, Suman Rawat and Jörg Bohlmann ∗ Biotechnology Laboratory, Department of Botany and Department of Forest Sciences, University of British Columbia, 6174 University Boulevard, Vancouver V6T 1Z3, B.C. Canada ( ∗ author for correspondence; e-mail: bohlmann@interchange.ubc.ca) Received 20 January 2002; accepted in revised form 21 May 2002 Key words: 3-carene, chemical plant defense, conifer biotechnology, methyl jasmonate, monoterpene synthase, Picea abies (Norway spruce) Abstract Picea abies (L.) Karst. (Norway spruce) employs constitutive and induced resin terpenoids as major chemical and physical defense-shields against insects and pathogens. In recent work, we showed that a suite of terpenoids, monoterpenoids and diterpenoids was induced in stems of Norway spruce after treatment of trees with methyl jasmonate (MeJA) (Martin et al., 2002). Increase of enzyme activities of terpenoid biosynthesis and accumulation of terpenoids was associated with MeJA-induced de novo differentiation of xylem resin ducts. The formation of defense-related traumatic resin ducts was also found in Norway spruce after attack by stem boring insects or after infestation with fungal pathogens. In the present study, we analyzed the traumatic resin response in Norway spruce further at the molecular genetic level. Treatment of trees with MeJA induced transient transcript accumulation of monoterpenoid synthases and diterpenoid synthases in stem tissues of Norway spruce. In screening for defense- related terpenoid synthase (TPS) genes from Norway spruce, a full-length monoterpenoid synthase cDNA, PaJF67, was isolated and the recombinant enzyme expressed in E. coli and functionally characterized in vitro. The cloned PaJF67 cDNA represents a new monoterpenoid synthase gene and the gene product was identified as 3-carene synthase. The enzyme encoded by PaJF67 forms stereospecifically (+)-3-carene (78% of total product) together with minor acyclic and cyclic monoterpenes, including the mechanistically closely related terpinolene (11% of total product). (+)-3-Carene is a characteristic monoterpene of constitutive and induced oleoresin defense of Norway spruce and other members of the Pinaceae. Abbreviations: 2D-GC, two-dimensional gas chromatography; GC, gas chromatography; GPP, geranyl diphospahte; GGPP, geranylgeranyl diphospahte; FPP, farnesyl diphosphate; LPP, linalyl diphosphate; MeJA, methyl jasmonate; MS, mass spectroscopy; TPS, terpenoid synthase. Introduction Conifer resins are composed of a large suite of struc- turally diverse terpenoid natural products that have long been recognized for their many functions in the chemical ecology of conifers and coniferophagus in- sects (Berryman, 1972; Langenheim, 1994; Bohlmann and Croteau, 1999; Seybold et al., 2000; Trapp and Croteau, 2001), as well as for their industrial value (Dawson, 1994; Schrader and Berger, 2001). The oleoresin of Norway spruce (Picea abies) con- sists mainly of monoterpenoids (10-carbon), sesquiter- penoids (15-carbon), and diterpenoids (20-carbon). Monoterpenoids and diterpenoids are equally abun- dant and together account for more than 98% of resin terpenoids in stems of Norway spruce (Martin et al., 2002). The turpentine fraction of Norway spruce resin includes more than 20 different monoterpenoids,