An integrated genomic, proteomic and biochemical analysis of (+)‐3‐carene biosynthesis in Sitka spruce (Picea sitchensis) genotypes that are resistant or susceptible to white pine weevil

Abstract

SUMMARYConifers are extremely long-lived plants that have evolved complex chemical defenses in the form of oleoresin terpenoids to resist attack from pathogens and herbivores. In these species, terpenoid diversity is determined by the size and composition of the terpene synthase (TPS) gene family and the single-and multi-product profiles of these enzymes. The monoterpene (+)-3-carene is associated with resistance of Sitka spruce (Picea sitchensis) to white pine weevil (Pissodes strobi). We used a combined genomic, proteomic and biochemical approach to analyze the (+)-3-carene phenotype in two contrasting Sitka spruce genotypes. Resistant trees produced significantly higher levels of (+)-3-carene than susceptible trees, in which only trace amounts were detected. Biosynthesis of (+)-3-carene is controlled, at the genome level, by a small family of closely related (+)-3-carene synthase (PsTPS-3car) genes (82-95% amino acid sequence identity). Transcript profiling identified one PsTPS-3car gene (PsTPS-3car1) that is expressed in both genotypes, one gene (PsTPS-3car2) that is expressed only in resistant trees, and one gene (PsTPS-3car3) that is expressed only in susceptible trees. The PsTPS-3car2 gene was not detected in genomic DNA of susceptible trees. Target-specific selected reaction monitoring confirmed this pattern of differential expression of members of the PsTPS-3car family at the proteome level. Kinetic characterization of the recombinant PsTPS-3car enzymes identified differences in the activities of PsTPS-3car2 and PsTPS-3car3 as a factor contributing to the different (+)-3-carene profiles of resistant and susceptible trees. In conclusion, variation of the (+)-3-carene phenotype is controlled by copy number variation of PsTPS-3car genes, variation of gene and protein expression, and variation in catalytic efficiencies.

Plasticity and Evolution of (+)-3-Carene Synthase and (−)-Sabinene Synthase Functions of a Sitka Spruce Monoterpene Synthase Gene Family Associated with Weevil Resistance

“…Cultures were then cooled to 16°C, induced by addition of isopropyl ␤-D-1-thiogalactopyranoside (final concentration 0.1 mM), and grown for 16 h at 180 rpm before harvesting. Recombinant protein was extracted and nickel affinity purified as previously described (7,11). Protein was quantified by BCA assay (Thermo Scientific) and SDS-PAGE with measurement of protein band intensity performed with the program ImageJ.…”

“…Resistance was positively associated with higher levels of the bicyclic monoterpene (ϩ)-3-carene (6). Subsequently, Hall et al (7) used a combination of genomic, target specific proteomic, and biochemical approaches to study the basis of variation of (ϩ)-3-carene levels in two contrasting genotypes of Sitka spruce, resistant genotype H898 trees with relatively high levels of (ϩ)-3-carene and susceptible genotype Q903 trees with trace levels of (ϩ)-3-carene. This work identified a small family of (ϩ)-3-carene synthase-like genes in Sitka spruce that contains the three (ϩ)-3-carene synthases PsTPS3car1, 3 PsTPS-3car2, PsTPS-3car3, and the (Ϫ)-sabinene synthase PsTPS-sab.…”

“…All four enzymes produce ␣-terpinolene as the second most abundant product plus a set of additional minor products (7) ( Table 1, supplemental Table S1). These similar traits and the particular presence of a (Ϫ)-sabinene synthase as a closely related enzyme with a group of (ϩ)-3-carene synthases suggested a pattern of divergent evolution in which PsTPS-sab arose from a PsTPS-3car ancestor through gene duplication and shift of function (7).…”

“…1). These four enzymes are multiproduct enzymes with the same overall product profile of monoterpenes, however, with different relative amounts of individual compounds (7). Most notably, PsTPS-3car1, PsTPS-3car2, and PsTPS-3car3 have (ϩ)-3-carene as the predominant product, whereas PsTPS-sab forms (Ϫ)-sabinene as the predominant product.…”

“…This work identified a small family of (ϩ)-3-carene synthase-like genes in Sitka spruce that contains the three (ϩ)-3-carene synthases PsTPS3car1, 3 PsTPS-3car2, PsTPS-3car3, and the (Ϫ)-sabinene synthase PsTPS-sab. Genotype-specific variations of gene copy number, transcript and protein expression, and catalytic efficiencies of members of this family are responsible for the difference in (ϩ)-3-carene levels (7). Specifically, the genomic presence, transcript and protein expression, and enzyme activity of PsTPS-3car2 accounted for much of the high levels of (ϩ)-3-carene in the resistant genotype.…”