Thomas J. SAVAGE and Rodney CROTEAU
Institute of Biological Chemistry. Washington State University, Pullman,
WA 99164-6340, USA
Conifer oleoresin, typically a mixture of volatile monoterpene hydrocarbons and nonvolatile diterpene resin acids, plays a major role in conifer-bark beetle interactions. Healthy trees accumulate copious amounts of oleoresin which can engulf and expell attacking beetles, while the insects exploit oleoresin monoterpenes as precursors for aggregation pheromone biosynthesis. Detailed characterization of the enzymology and molecular biology of oleoresin biosynthesis is essential for understanding the physiology of conifer defense and for elucidating the underlying mechanisms of conifer-bark beetle coevolution.
Monoterpenes are derived from lhe C10allylic pyrophosphate geranyl diphosphate in a reaction catalyzed hy monoterpene synthases (or "cyclases" because of the cyclic nature of most hydrocarbon products). Analysis of monoterpene synthase activity in wounded and unwounded saplings of ten North American conifer species suggests that conifers vary in levels of constitutive and wound-inducible monoterpene biosynthesis. Species such as lodgepole pine (Pinus contorta), a conitcr species native to western North America and a major host of the mountain pine beetle (Dendroctomus ponderosae), have constitutively high levels of monoterpene synthase activity which are largely unaffected by wounding. Four different monoterpene synthases have been isolated from lodgepole pine xylem, each catalyzing the conversion of geranyl diphosphate to multiple stereochemically-related monoterpene isomers found in lodgepole pine oleoresin-(3,4). All four enzymes are similar to each other in molecular weight, mono- and divalent metal ion requirement, pH optimum, and reactivity toward chemical modification reagents, but they have quite different properties from the analogous enzymes found in herbaceous angiosperm specics. One of the enzymes from lodgepole pine, (-)-phellandrene synthase, has been purified and identified as a 67 kD monoimeric protein.
In contrast, monoterpene synthase activity in stems of grand fir (Abies grandis), another western North American conifer which is host to the fir engraver beetle (Scolytus ventralis) is constitutively low but increases dramatically 7-14 days after wounding (1,2). Different sets of monoterpene synthase activities were isolated from wounded and unwounded saplings, indicating that the wound-induction of monoterpene synthase activity is the result of the novel expression of monoterpene synthases not found in unwounded tissue (5). One of these wound-inducible enzymes, (-)-pinene synthase, has been purified to homogeneity and is similar in characteristics to the monoterpene synthases of lodgepole pine (6). Probing blots of proteins extracted from wounded and unwounded trees with polyclonal antibodies raised against the purified enzyme revealed that the wound-induction of monoterpene synthase activity was correlated with increases in antigenic cyclase protein, suggesting that enzyme activity levels are regulated at thc translational or transcriptional levels (7). Genes encoding monoterpene synthases of grand fir have recently heen isolated, and current efforts are directed towards understanding the molecular events controlling monoterpene synthase gene expression.
Non-volatile diterpene resin acids arise from the oxidation of diterpene hydrocarbons generated from the C20 allylic pyrophosphate geranylgeranyl diphosphate in a reaction catalyzed by diterpene syntheses (or cyclases). Abietadiene synthase, the enzyme responsible for catalyzing the conversion of geranylgeranyl diphosphate to the abieticacid precursor abietadiene, has been detected in both lodgepole pine and grand fir (8), and was purified to homogeneity from cell-free extracts of wounded grand fir stems. Amino acid sequences of proteolytic fragments from the purified enzyme were used to design degenerate primers for the polymerase chain reaction amplification of a fragment of abietadiene synthase cDNA, which was subsequently used to isolate a full-length cDNA clone encoding the enzyme. Abietadiene and abietadienol hydroxylase activities, responsible for the oxidation of abietadiene to abietadienol and the subsequent oxidation of the alcohol to abietadienal, respectively, have been detected in microsomal preparations from cell-free extracts of both lodgepole pine and grand fir (9). The final enzymatic step involved in abietic acid biosynthesis, the oxidation of abietadienal to the corresponding acid, was also demonstrated in soluble fractions of extracts from both species. Interestingly, in wounded grand fir tissue, all enzymatic activities except the terminal dehydrogenase were coordinately induced along with monoterpene synthase activity, suggesting that wound stimulation of oleoresin biosynthesis involves translational or transcriptional expression of multiple genes encoding terpenoid pathway enzymes (l0).
While mono- and diterpenes are the major constituents of most conifer oleoresins, C11 alkanes are found as components of oleoresins of at least twelve different Pinus species native to western North America. Short-chain alkanes make up over 95% of cortical oleoresin volatiles in Pinus sabiniana and P. jeffreyi, two pine species that are resistant to bark beetle species which readily attack pine species containing monoterpenoid turpentine. We have initiated studies on the biosynthesis of short-chain alkanes (principally n-heptane) in P. jeffreyi because of their likely role in establishing the host specificity of bark beetle-conifer interactions in this species. Alkane biosynthesis in P. jeffreyi is highly tissue-specific; alkanes accumulate in xylem and phloem tissues but are not found in foliar tissue (11). Enamination of the effects of fatty acid synthase inhibitors, aldehyde trapping agents, and decarbonylation inhibitors on the incorporation of 14C into n-heptane and C8 intermediates in P. jeffreyi xylem incubated with 14C acetate suggests that n-heptane arises via an elongation/reduction/C1-elimination pathway. Current research efforts are directed towards detecting specific enzymatic activities involved in short-chain alkane biosynthesis.
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