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.
Diterpene resin acids (DRAs) are major components of pine (Pinus spp.) oleoresin. They play critical roles in conifer defense against insects and pathogens and as a renewable resource for industrial bioproducts. The core structures of DRAs are formed in secondary (i.e. specialized) metabolism via cycloisomerization of geranylgeranyl diphosphate (GGPP) by diterpene synthases (diTPSs). Previously described gymnosperm diTPSs of DRA biosynthesis are bifunctional enzymes that catalyze the initial bicyclization of GGPP followed by rearrangement of a (+)-copalyl diphosphate intermediate at two discrete class II and class I active sites. In contrast, similar diterpenes of gibberellin primary (i.e. general) metabolism are produced by the consecutive activity of two monofunctional class II and class I diTPSs. Using high-throughput transcriptome sequencing, we discovered 11 diTPS from jack pine (Pinus banksiana) and lodgepole pine (Pinus contorta). Three of these were orthologous to known conifer bifunctional levopimaradiene/abietadiene synthases. Surprisingly, two sets of orthologous PbdiTPSs and PcdiTPSs were monofunctional class I enzymes that lacked functional class II active sites and converted (+)-copalyl diphosphate, but not GGPP, into isopimaradiene and pimaradiene as major products. Diterpene profiles and transcriptome sequences of lodgepole pine and jack pine are consistent with roles for these diTPSs in DRA biosynthesis. The monofunctional class I diTPSs of DRA biosynthesis form a new clade within the gymnosperm-specific TPS-d3 subfamily that evolved from bifunctional diTPS rather than monofunctional enzymes (TPS-c and TPS-e) of gibberellin metabolism. Homology modeling suggested alterations in the class I active site that may have contributed to their functional specialization relative to other conifer diTPSs.Conifer trees, including lodgepole pine (Pinus contorta) and jack pine (Pinus banksiana), produce complex mixtures of mono-, sesqui-, and diterpenoid specialized (i.e. secondary) metabolites, most prominently in the form of oleoresin, that can act as a physical and chemical defense against insect and pathogen attack
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