Myrcene Hydroxylases do not Determine Enantiomeric Composition of Pheromonal Ipsdienol in Ips spp.
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Myrcene (7-methyl-3-methylene-1,6-octadiene) hydroxylation is likely one of the final reactions involved in the production of the Ips spp. (Coleoptera: Scolytidae) aggregation pheromone components, ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) and ipsenol (2-methyl-6-methylene-7-octen-4-ol). To gain insight into the evolution of pheromone production, we isolated a full-length cDNA from the pinyon ips, Ips confusus (LeConte), that encodes a pheromone-biosynthetic cytochrome P450, I. confusus CYP9T1 (IcCYP9T1). The recovered cDNA is 1.70 kb, and the open reading frame encodes a 532 amino acid protein. IcCYP9T1 is 94% identical to the pine engraver, Ips pini (Say), CYP9T2 ortholog that hydroxylates myrcene. Quantitative real-time PCR experiments showed that IcCYP9T1, as does CYP9T2, has an expression pattern similar to other pheromone-biosynthetic genes in I. pini. Basal expression levels were higher in males than females, and expression was significantly induced in male, but not in female, anterior midguts by feeding on host phloem. Microsomes, prepared from Sf9 cells co-expressing baculoviral-mediated recombinant IcCYP9T1 and house fly (Musca domestica) NADPH-cytochrome P450 reductase, converted myrcene to ~85%-(R)-(−)-ipsdienol. These results are consistent with IcCYP9T1 encoding a myrcene hydroxylase that functions near the end of the pheromone-biosynthetic pathway. Since the I. confusus pheromone blend contains >90%-(S)-(+)-ipsdienol, these results confirm further that Ips spp. myrcene hydroxylases do not control the final ipsdienol enantiomeric blend. Other enzymes are required following myrcene hydroxylation to achieve the critical quantity and enantiomeric composition of pheromonal ipsenol and ipsdienol used by different Ips spp.
KeywordsIps Bark beetle Pheromone P450 Pheromone biosynthesis Monoterpene Functional expression
We thank D. Vanderwel for deuterium-labeled myrcene and for sharing unpublished data, M. Schuler for the housefly P450 reductase baculoviral clone, C. Oehlschlager for the ipsdienol standard, D. Nelson for the naming of Ips confusus CYP9T1 in accordance with current P450 nomenclature, the Nevada Genomics Center for assistance with sequencing and qRT-PCR, D. Quilici at the Nevada Proteomics Center for GC-MS analysis, H. Damke for help with baculovirus expression, A. Griffith for studies of potential housekeeping genes, other members of the laboratories of GJB and CT for assistance with collecting beetles, dissections, assays, and helpful advice, and the Bureau of Land Management and US Forest Service for permission to collect beetle-infested trees. This work was supported by USDA-NRI (2006-35604-16727), NSF (IBN 0316370), and a HATCH grant from the Nevada Agriculture Experiment Station (NAES) (NEV00339). This paper is a contribution of the NAES, publication # 03087107.
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