We analyzed the expression pattern of various terpene synthase (TPS) genes in response to a wounding injury applied to the apical leader of Sitka spruce (Picea sitchensis Bong. Carr.) genotypes known to be resistant (R) or susceptible (S) to white pine weevil (Pissodes strobi Peck.) attack. The purpose was to test if differences in constitutive or wound-induced TPS expression can be associated with established weevil resistance. All wounding treatments were conducted on 9-year-old R and S trees growing under natural field conditions within the range of variation for weevil R and S genotypes. Representative cDNAs of the monoterpene synthase (mono-TPS), sesquiterpene synthase (sesqui-TPS), and diterpene synthase (di-TPS) classes were isolated from Sitka spruce to assess TPS transcript levels. Based on amino acid sequence similarity, the cDNAs resemble Norway spruce (Picea abies) (2)-linalool synthase (mono-TPS; PsTPS-Linl) and levopimaradiene/abietadiene synthase (di-TPS; PsTPS-LASl), and grand fir (Abies grandis) d-selinene synthase (sesqui-TPS; PsTPS-Sell). One other mono-TPS was functionally identified as (2)-limonene synthase (PsTPS-Lim). No significant difference in constitutive expression levels for these TPSs was detected between R and S trees. However, over a postwounding period of 16 d, only R trees exhibited significant transcript accumulation for the mono-and sesqui-TPS tested. Both R and S trees exhibited a significant accumulation of PsTPS-LASl transcripts. An assessment of traumatic resin duct formation in wounded leaders showed that both R and S trees responded by forming traumatic resin ducts; however, the magnitude of this response was significantly greater in R trees. Collectively, our data imply that the induced resinosis response is an important aspect of defense in weevil R Sitka spruce trees growing under natural conditions.

Large-scale sequencing of conifer cDNAs and targeted molecular cloning have identified many putative conifer defense genes. Expression of many of these genes is induced in response to biotic stress and some may be expressed only in a few specialized tissues or cells. Proven functional genomics approaches to test these genes involve expression of proteins in Escherichia coli or yeast for biochemical characterization or constitutive over-expression in transformed plants. Plant transformation to test functions of insect-, wound- or pathogen-induced conifer defense genes would benefit from the use of an inducible expression system. We describe here the development of a heterologous, wound- and insect-inducible gene expression system for conifers using the potato proteinase inhibitor II (pinII)-promoter. Using pinII::GUS and pinII::(E)-alpha-bisabolene synthase expression constructs we demonstrate localized induced gene expression in white spruce seedlings (Picea glauca). Testing of these constructs in Arabidopsis thaliana and tobacco illustrates the additional potential of the pinII-promoter to be used in tests of gene function that involve cell-specific and systemic induction.