Grand fir (Abies grandis) has been developed as a model system for the study of oleoresin production in response to stem wounding and insect attack. The
turpentine fraction of the oleoresin was shown to contain at least 38
sesquiterpenes that represent 12.5% of the
turpentine, with the
monoterpenes comprising the remainder. Assays of cell-free extracts from grand fir stem with
farnesyl diphosphate as substrate indicated that the constitutive
sesquiterpene synthases produced the same
sesquiterpenes found in the oleoresin and that, in response to wounding, only two new products were synthesized,
delta-cadinene and (E)-alpha-bisabolene. A similarity based cloning strategy yielded two new
cDNA species from a stem cDNA library that, when expressed in Escherichia coli and the gene products subsequently assayed, yielded a remarkable number of
sesquiterpene products. The encoded
enzymes have been named
delta-selinene synthase and
gamma-humulene synthase based on the principal products formed; however, each
enzyme synthesizes three major products and produces 34 and 52 total
sesquiterpenes, respectively, thereby accounting for many of the
sesquiterpenes of the oleoresin. The deduced amino acid sequence of the
delta-selinene synthase cDNA open reading frame encodes a
protein of 581 residues (at 67.6 kDa), whereas that of the
gamma-humulene synthase cDNA encodes a
protein of 593 residues (at 67.9 kDa). The two amino acid sequences are 83% similar and 65% identical to each other and range in similarity from 65 to 67% and in identity from 43 to 46% when compared with the known sequences of
monoterpene and
diterpene synthases from grand fir. Although the two
sesquiterpene synthases from this gymnosperm do not very closely resemble
terpene synthases from angiosperm species (52-56% similarity and 26-30% identity, there are clustered regions of significant apparent homology between the
enzymes of these two plant classes. The multi-step, multi-product reactions catalyzed by the
sesquiterpene synthases from grand fir are among the most complex of any
terpenoid cyclase thus far described.