Grand fir (Abies grandis) has been developed as a model system for studying 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 (C10)
olefins in which (-)-
limonene and (-)-alpha- and (-)-
beta-pinene are prominent components; (-)-
limonene and (-)-pinene synthase activities are also induced upon stem wounding. A similarity based cloning strategy yielded three new
cDNA species from a wounded stem cDNA library that appeared to encode three distinct
monoterpene synthases. After expression in Escherichia coli and
enzyme assay with
geranyl diphosphate as substrate, subsequent analysis of the
terpene products by chiral phase gas chromatography and mass spectrometry showed that these sequences encoded a (-)-
limonene synthase, a
myrcene synthase, and a (-)-pinene synthase that produces both
alpha-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 sequences 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 comparison revealed that these
monoterpene synthases from grand fir resemble
sesquiterpene (C15) synthases and
diterpene (C20) synthases from conifers more closely than other
monoterpene synthases from angiosperm species. This similarity between extant
monoterpene,
sesquiterpene, and
diterpene synthases of gymnosperms is surprising since functional diversification of this
enzyme class is assumed to have occurred over 300 million years ago.
Wound-induced accumulation of transcripts for
monoterpene synthases was demonstrated by
RNA blot hybridization using probes derived from the three
monoterpene synthase cDNAs. The availability of
cDNA species encoding these
monoterpene synthases will allow an understanding of the regulation of oleoresin formation in conifers and will ultimately permit the transgenic manipulation of this defensive secretion to enhance resistance to insects. These cDNAs also furnish tools for defining structure-function relationships in this group of catalysts that generate acyclic, monocyclic, and bicyclic
olefin products.