Pan-genome analysis of soybean terpene synthase identifies GmTPS20 as a defense-related linalool synthase

Soybean terpene synthase (TPS) genes are pivotal to ecological adaptation and stress resilience, yet their genetic diversity, evolutionary history, and enzymatic mechanisms remain poorly understood. A pan-genomic survey of 27 soybean genomes was performed to identify and classify TPS loci by conservation status. Phylogenetic reconstruction and Ka/Ks analysis were used to infer evolutionary relationships and selection regimes. An uncharacterized core gene, GmTPS20, and its close homolog GmTPS15 were prioritized for functional characterization using expression profiling, subcellular localization, in vitro enzyme assays with geranyl diphosphate (GPP), neryl diphosphate (NPP), and farnesyl diphosphate (FPP) isomers, transient expression in Nicotiana benthamiana, and structural docking. The pan-genome survey identified 26 TPS loci: 15 core, four near-core, five variable, and two private, highlighting strong purifying selection on conserved members alongside lineage-specific losses. Soybean TPSs fell within the TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g clades, with most loci exhibiting Ka/Ks < 1. GmTPS20 showed broad expression peaking in young leaves and was induced by insect herbivory and methyl jasmonate, whereas GmTPS15 was enriched in reproductive tissues. Both proteins localized to chloroplasts, consistent with the MEP pathway. GmTPS20 acted as a substrate-specific monoterpene synthase that converted GPP to linalool and NPP to linalool and nerol, but did not accept FPP isomers; transient expression in N. benthamiana confirmed linalool accumulation in planta. Under matched conditions, GmTPS15 produced no detectable volatile products. Structural docking indicated that both enzymes can bind GPP; however, GmTPS20 features a more compact diphosphate-coordination network and a deeper, narrower active site, whereas GmTPS15 adopts a more open pocket with reduced polar constraints, rationalizing their divergent catalytic behaviors. Collectively, these findings clarify mechanisms of TPS functional diversification in legumes and provide molecular targets for engineering terpenoid-based defense and desirable agronomic traits in soybean.