Significance: Whether insect sesquiterpenes are synthesized de novo, derived from plant precursors, or produced by symbionts is often unknown. We identified an evolutionarily novel terpene synthase gene family in the striped flea beetle, a notorious pest of Brassica crops in North America and Asia, and one of these genes was shown to be directly involved in the biosynthesis of the male-specific sesquiterpene aggregation pheromone. Phylogenetic and gene structure analyses indicate that an expansion of the trans -isoprenyl diphosphate synthase gene family in the ancestor of the subfamily Galerucinae enabled functional diversification toward this terpene synthase gene family. These insights into how flea beetles synthesize their aggregation pheromones may lead to new approaches for pest management.

Isoprenyl diphosphate synthases (IDSs) produce the ubiquitous branched-chain diphosphates of different lengths that are precursors of all major classes of terpenes. Typically, individual short-chain IDSs (scIDSs) make the C ₁₀ , C ₁₅ , and C ₂₀ isoprenyl diphosphates separately. Here, we report that the product length synthesized by a single scIDS shifts depending on the divalent metal cofactor present. This previously undescribed mechanism of carbon chain-length determination was discovered for a scIDS from juvenile horseradish leaf beetles, Phaedon cochleariae . The recombinant enzyme P. cochleariae isoprenyl diphosphate synthase 1 ( Pc IDS1) yields 96% C ₁₀ -geranyl diphosphate (GDP) and only 4% C ₁₅ -farnesyl diphosphate (FDP) in the presence of Co ²⁺ or Mn ²⁺ as a cofactor, whereas it yields only 18% C ₁₀ GDP but 82% C ₁₅ FDP in the presence of Mg ²⁺ . In reaction with Co ²⁺ , Pc IDS1 has a K _m of 11.6 μM for dimethylallyl diphosphate as a cosubstrate and 24.3 μM for GDP. However, with Mg ²⁺ , Pc IDS1 has a K _m of 1.18 μM for GDP, suggesting that this substrate is favored by the enzyme under such conditions. RNAi targeting Pc IDS1 revealed the participation of this enzyme in the de novo synthesis of defensive monoterpenoids in the beetle larvae. As an FDP synthase, Pc IDS1 could be associated with the formation of sesquiterpenes, such as juvenile hormones. Detection of Co ²⁺ , Mn ²⁺ , or Mg ²⁺ in the beetle larvae suggests flux control into C ₁₀ vs. C ₁₅ isoprenoids could be accomplished by these ions in vivo. The dependence of product chain length of scIDSs on metal cofactor identity introduces an additional regulation for these branch point enzymes of terpene metabolism.

Norway spruce (Picea abies) defends itself against herbivores and pathogens by formation of traumatic resin ducts filled with terpenoid-based oleoresin. An important group of enzymes in terpenoid biosynthesis are the short-chain isoprenyl diphosphate synthases which produce geranyl diphosphate (C₁₀), farnesyl diphosphate (C₁₅), and geranylgeranyl diphosphate (C₂₀) as precursors of monoterpenes, sesquiterpenes, and diterpene resin acids, respectively. After treatment with methyl jasmonate (MJ) we investigated the expression of all isoprenyl diphosphate synthase genes characterized to date from Norway spruce and correlated this with formation of traumatic resin ducts and terpene accumulation. Formation of traumatic resin ducts correlated with higher amounts of monoterpenes, sesquiterpenes and diterpene resin acids and an upregulation of isoprenyl diphosphate synthase genes producing geranyl diphosphate or geranylgeranyl diphosphate. Among defense hormones, jasmonate and jasmonate-isoleucine conjugate accumulated to higher levels in trees with extensive traumatic resin duct formation, whereas salicylate did not. Jasmonate and ethylene are likely to both be involved in formation of traumatic resin ducts based on elevated transcripts of genes encoding lipoxygenase and 1-aminocyclopropane-1-carboxylic acid oxidase associated with resin duct formation. Other genes involved in defense signalling in other systems, mitogen-activated protein kinase3 and nonexpressor of pathogenesis-related gene1, were also associated with traumatic resin duct formation. These responses were detected not only at the site of MJ treatment, but also systemically up to 60 cm above the site of treatment on the trunk.