Biochemistry of Oleoresinosis

Abstract

ABSTRACI Elevated levels of monoterpenes and diterpene resin acids are produced in the stems of lodgepole pine (Pinus contorta var latifolia) saplings when wounded and inoculated with the blue-stain fungus Ceratocystis clavigera or when wounded and treated with a pectic fragment from tomato leaves (PIIF) or a fungal cell wall fragment (chitosan). This induced defensive response (hyperoleoresinosis) is the result ofa transient rise in the ability to biosynthesize cyclic monoterpenes and diterpene resin acids as measured by the in vivo incorporation of label from [U-'4Clsucrose relative to untreated controls, and is accompanied by a corresponding rise in the levels or activities of the relevant terpene cyclases as determined by in vitro assay using labeled acycic precursors. The results indicate that juvenile P. contorta responds to infection and biotic elicitors much like the mature tree, and they suggest that the Pinaceae possess a mechanism for elicitor recognition and induced defense similar to that of other higher plants. 25, 26) and the broad outlines of pine defense biology have been delineated (2, 15). The biosynthesis of resin terpenoids, however, is poorly understood, as is the biochemistry of the defense reaction; present concepts are based largely on analogy to terpen-oid metabolism and phytoalexin production in herbaceous species (7, 15, 17). The lack of fundamental information about this defense response is, in part, a result ofthe difficulties encountered in experimenting with mature trees in the forest setting, the system in which the phenomenon was first discovered and with which most early observations were made (17, 19, 21, 23, 24). In this communication we provide an account of induced oleo-resinosis in 2-year-old lodgepole pine saplings (maintained in a greenhouse) in response to inoculation with 'blue staining' fungus (C. clavigera) and with carbohydrate elicitors, we report in vivo experiments-which demonstrate the elevated formation of mon-oterpenes and diterpene resin acids, and we describe the isolation and enhanced activity of the terpenoid cyclases which catalyze the committed steps of monoterpene and diterpene biosynthesis in this species. The terpenoid fraction ofpine resin contains 20 to 50% volatile monoterpenes (C10), minor levels of sesquiterpenoids (C5), and 50 to 80% nonvolatile diterpene acids (C20) (8, 18), and the induced secretion of these materials constitutes an important component of the defense response of pines to attack by bark beetles and their associated pathogenic fungi (2, 13, 26). Resistant lodgepole pine (Pinus contorta Douglas) produces high levels of a-pinene and limonene (Fig. 1) in the secondary resin elicited by attack (relative to the constitutive [preformed] resin contained in the resin ducts) (19); however, the major factor in resistance to the mountain pine beetle (Dendroctonus ponderosae Hopkins) and associated fungus (Ceratocystis clavigera Robinson et Dav-idson) appears to be a rapid and vigorous secondary resinosis resulting from the increased production of all constitutive ter-penoids (21, 23). Mature lodgepole pine inoculated with C. clavigera, a pectic fragment from plant cell walls, or a fungal cell wall fragment (chitosan) respond by producing resin monoter-penes to the same or greater extent as control trees in which the response is elicited by living mountain pine beetle, suggesting that pines, like other higher plants, possess a common recognition defense mechanism (17). The chemistry of pine resin and the role of resin components in resistance to infestation and infection have been discussed (20, '