MONOTERPENE SYNTHASES FROM GRAND FIR (ABIES-GRANDIS) - CDNA ISOLATION, CHARACTERIZATION, AND FUNCTIONAL EXPRESSION OF MYRCENE SYNTHASE, (-)(4S)-LIMONENE SYNTHASE, AND (-)-(1S,5S)-PINENE SYNTHASE

Grand fir (Abies grandis) has been developed as a model system for stu dying defensive oleoresin formation in conifers in response to insect attack or other injury, The turpentine fraction of the oleoresin is a complex mixture of monoterpene (C-10) olefins in which (-)-limonene an d (-)-alpha- and (-)-beta-pinene are prominent components; (-)-limonen e and (-)-pinene synthase activities are also induced upon stem woundi ng, A similarity based cloning strategy yielded three new cDNA species from a mounded stem cDNA library that appeared to encode three distin ct monoterpene synthases, After expression in Escherichia coli and enz yme assay with geranyl diphosphate as substrate, subsequent analysis o f the terpene products by chiral phase gas chromatography and mass spe ctrometry showed that these sequences encoded a (-)-limonene synthase, a myrcene synthase, and a (-)-pinene synthase that produces both alph a-pinene and beta-pinene. In properties and reaction stereochemistry, the recombinant enzymes resemble the corresponding native monoterpene synthases of wound-induced grand fir stem. The deduced amino acid sequ ences indicated the limonene synthase to be 637 residues in length (73 .5 kDa), the myrcene synthase to be 627 residues in length (72.5 kDa), and the pinene synthase to be 628 residues in length (71.5 kDa); all of these monoterpene synthases appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence. Sequence compari son revealed that these monoterpene synthases from grand fir resemble sesquiterpene (C-15) synthases and diterpene (C-20) synthases from con ifers more closely than other monoterpene synthases from angiosperm sp ecies. This similarity between extant monoterpene, sesquiterpene, and diterpene synthases of gymnosperms is surprising since functional dive rsification of this enzyme class is assumed to have occurred over 300 million years ago. Wound-induced accumulation of transcripts for monot erpene synthases was demonstrated by RNA blot hybridization using prob es derived from the three monoterpene synthase cDNAs. The availability of cDNA species encoding these monoterpene synthases mill allow an un derstanding of the regulation of oleoresin formation in conifers and w ill ultimately permit the transgenic manipulation of this defensive se cretion to enhance resistance to insects. These cDNAs also furnish too ls for defining structure-function relationships in this group of cata lysts that generate acyclic, monocyclic, and bicyclic olefin products.