Terpene synthase diversity reveals the inter- and intraspecific floral scent evolution in Aquilegia

Floral volatile terpenes play pivotal roles in plant-pollinator interactions and ecological adaptation, yet the genetic mechanisms underlying their diversification in Aquilegia remain poorly understood. This study integrates gas chromatography-mass spectrometry (GC-MS) and functional characterization assays to investigate how terpene synthase (TPS) diversity shapes floral scent evolution across Aquilegia species and populations. We identified (+)-limonene, linalool, β-pinene, and β-sesquiphellandrene as the dominant floral terpenes, with substantial inter- and intraspecific variation driven by differential expression and sequence divergence of key TPS genes, including TPS7, TPS8, TPS9, and TPS24. Functional characterization revealed that orthologous variants of TPS7 and TPS8 influence species-specific monoterpene and sesquiterpene production, while TPS24 variants exhibit substrate plasticity in linalool synthesis despite retaining conserved catalytic motifs. Population-level analyses of Aquilegia viridiflora revealed that frame-shift mutations and expression shifts in TPSs are associated with terpene profile divergence, while residue variations in allelic TPS variants may have driven functional novelty during evolution. Our findings highlight TPS diversity as a molecular mechanism for floral scent diversification, enabling adaptive radiation through pollinator-mediated selection and specialization within distinct ecological niches. This study provides crucial insights into the evolutionary interplay between genetic variation, enzymatic plasticity, and ecological adaptation in shaping plant biodiversity.