Karrikins are abiotic signals found in bushfire smoke that enhance germination and seedling responses to light. Karrikins are structurally similar to strigolactones, phytohormones that regulate numerous processes, including shoot branching and colonisation of roots by arbuscular mycorrhizal fungi.
KAI2 and D14 are two homologous α/β-hydrolase enzymes that are also receptors for karrikins and strigolactones respectively. Both proteins are highly conserved across seed plants, and share an essential catalytic triad in a similar structural orientation, which is necessary for both ligand hydrolysis and signalling to occur.
Evolutionary evidence suggests that D14, and therefore strigolactone signalling in seed plants, has emerged through duplication and neofunctionalisation of KAI2. This raises questions on how ligand specificity of these two proteins might have evolved following divergence from a common ancestor.
Ancestral sequence reconstruction (ASR) is a method of generating hypothetical ancestral sequences based on the sequences of existing relatives and their phylogenetic relationships. Here, we report data on recombinant ancestral reconstructions of both KAI2 and D14, including crystal structures, functional assays, and biological complementation studies. We show that ancestral KAI2 and D14 display novel ligand specificity at the biochemical level, and have partial biological activity when expressed in Arabidopsis thaliana. These results are consistent with a model in which the specificity of ligand binding and downstream signalling has evolved shortly after the duplication of KAI2.