Tuckey_2026_Plant.J_127_e71113

Strigolactones are phytohormones that regulate shoot branching and facilitate communication with neighbouring plants, bacteria and arbuscular mycorrhizal fungi. In seed plants, strigolactone perception begins with the enzyme-receptor DWARF14 (D14), an alpha/beta-hydrolase likely to have evolved via gene duplication within a larger gene family that includes the karrikin receptor, KARRIKIN INSENSITIVE 2 (KAI2) and other D14-like (DLK) proteins of uncertain function. However, it is unclear when the functional characteristics that define a bona fide strigolactone receptor evolved. Here, we apply ancestral sequence reconstruction to generate a D14 protein representative of seed plants to study the evolution of ligand specificity and the conservation of signalling mechanisms and to explore desirable traits for protein engineering. Ancestrally reconstructed D14 is structurally and functionally comparable to D14 from Arabidopsis thaliana. The ligand specificity of ancestral D14 does not meaningfully differ, despite having unusual active site geometry and altered enzyme kinetics. We show that the catalytic aspartic acid residue of ancestral D14 is not required for strigolactone signalling when expressed in A. thaliana, suggesting that this mechanistic feature is conserved throughout seed plants. We find that ancestral D14 exhibits higher recombinant yields, greatly increased thermostability and enhanced catalytic activity relative to D14 from A. thaliana. This work provides insight into the evolution of phytohormone signalling and presents a robust scaffold for the application of D14-type proteins in diverse applications.