OPP GPP a - Pinene p - Pinene 3 - Carene p - Phellandrene Limonene I , - ' lPiee - inn

Grand fir (Abies grandis) saplings and derived cell cultures are useful systems for studying the regulation of defensive oleoresinosis in conifers, a process involving both the constitutive accumulation of resin (pitch) in specialized secretory structures and the induced production of monoterpene olefins (turpentine) and diterpene resin acids (rosin) by nonspecialized cells at the site of injury. The pathways and enzymes involved in monoterpene and diterpene resin acid biosynthesis are described, as are the coinduction kinetics following stem injury as determined by resin analysis, enzyme activity measurements, and immunoblotting. The effects of seasonal development, light deprivation, and water stress on constitutive and wound-induced oleoresinosis are reported. Future efforts, including a PCR-based cloning strategy, to define signal transduction in the wound response and the resulting gene activation processes are delineated. Many conifer species respond to bark beetle attack by secreting oleoresin (pitch) at the wound site (1, 2). This oleoresin, composed of roughly equal quantities of monoterpenes (turpentine) and diterpenoid resin acids (rosin) (3), is toxic to both beetles and their pathogenic fungal associates (4), and, after evaporation of the volatile turpentine, it forms a hardened rosin barrier to seal the injury (5). Interestingly, many species of bark beetles are specifically attracted to the monoterpene compounds emitted from their conifer hosts and can utilize components of the host oleoresin in the synthesis of pheromones for promoting aggregation (6). These same volatile products may also serve as attractants for beetle predators and parasitoids. Thus, the chemical ecological relationships between conifer host, beetle pest, and beetle predator are exceedingly complex and present several possible avenues for manipulating oleoresin composition to improve tree resistance, for example, by disguising the host, or altering the levels of pheromone precursors or predator attractants (6). Constitutive oleoresin is synthesized in the epithelial cells of specialized secretory structures such as stem resin blisters or ducts in which the oleoresin is accumulated, whereas induced oleoresin appears to originate in nonspecialized cells, adjacent to the site of injury, that are not normally associated with such large-scale production of terpenoids (7-9). That both constitutive and localized, inducible resin-based defense mechanisms seem to have been selected for in this group of plants is most unusual. However, the major conifer types [pines (Pinus), spruces (Picea), larches (Larix), and true firs (Abies)] do differ considerably in their apparent reliance on constitutive or induced resin defenses (10). Oleoresinosis in species of pines, 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. which accumulate large quantities of stored oleoresin in extensive duct systems, is not highly elevated in response to injury, whereas in true firs, which accumulate relatively little constitutive material in resin blisters, the rate of oleoresin production increases manyfold upon challenge (10, 11). In both constitutive and induced defenses, the analytical and microscopic evidence (12, 13) suggests that the monoterpenes and diterpenoids are formed coincidentally in the same locale, consistent with the requirement for an organic solvent (turpentine) in which the water-insoluble diterpene resin acids can be dissolved and translocated (5). Oleoresin Biochemistry and Enzymology Grand fir (Abies grandis Lindl.) has proved to be a very useful model for the study of inducJd oleoresin production (6, 14). Formation of oleoresin monoterpenes in this, and related, conifer species is catalyzed by a series of synthases (cyclases) that transform the common C1o isoprenoid precursor geranyl pyrophosphate to olefins of the various skeletal types (Fig. 1) (11, 15). The increase in monoterpene biosynthesis after wounding of grand fir stem is the result of the apparent enhancement of constitutive activities (principally limonene synthase) and the appearance of distinct, inducible activities (including 3-carene, P-phellandrene, and aand 3-pinene synthases) (15). The enzymes responsible for constitutive turpentine production are very similar in general properties to the inducible forms; however, these gymnosperm monoterpene cyclases are distinguishable in several characteristics (pH optimum, metal ion requirement) from their angiosperm counterparts (16), which they nevertheless resemble in mechanism of action (17). The principal wound-inducible monoterpene cyclase of grand fir stem produces both (-)-a-pinene and (-)-p3-pinene, in a fixed 2:3 ratio, from geranyl pyrophosphate via a common cationic intermediate (18). This most unusual catalytic feature of multiple product formation is, in fact, fairly common among the monoterpene cyclases (19, 20), and it was confirmed in the case of the (-)-pinene synthase by exploiting the phenomenon of isotopically sensitive branching with substrates bearing strategically placed deuterium atoms (21). This 62-kDa monomeric enzyme has been purified to apparent homogeneity, characterized in some detail, and used to raise polyclonal antibodies in rabbits (18, 22). Western immunoblot analysis with the anti-pinene synthase granuloma antiserum indicated strong cross-reactivity with all of the monoterpene cyclases from grand fir but no detectable recognition of any cyclase from pine, spruce, or other conifer genera (22). Thus, this pinene synthase is more closely related to other cyclases of fir *To whom reprint requests should be addressed.