Conservation of IAMT preference for indole acetic acid methylation across 250 million years of seed plant divergence, with only one recent evolutionary switch in <i>Ocimum</i>

Abstract Plant indole-3-acetic acid methyltransferase (IAMT) is an ancient SABATH enzyme that modulates auxin levels via S-adenosyl-L-Methionine (SAM)-dependent methylation. While most IAMTs studied previously prefer to methylate indole-3-acetic acid (IAA), one orthologous enzyme prefers to methylate cinnamic acid (CA) and shows no activity towards IAA. To understand whether other closely related enzymes also show substrate preference switches, we combined molecular phylogenetic analyses with in-vitro enzyme assays. A maximum-likelihood tree of 704 IAMT-like enzymes shows pervasive retention of IAMT orthologs across nearly all angiosperms and gymnosperms. Our newly reported enzymatic assays of 59 orthologs spanning 52 species and 26 orders revealed strongly conserved methylation preference for IAA across all lineages. However, in the genus Ocimum (Lamiaceae), species appear to possess one copy of IAMT that encodes an IAA-preferring enzyme and a second copy that encodes an enzyme that prefers to methylate CA. Altogether, we experimentally investigated four lineages, Lamiales, Solanales, Fabales and Rosales, in which IAMT-type enzymes were duplicated but no others showed evidence for substrate preference switches. Alignments and computational modeling of Lamiaceae sequences highlighted M35, L325, and L363 as amino acid substitutions that may account for CA preference evolution in Ocimum enzymes. Yet, experimental mutation of those sites in either modern-day or ancestral sequences did not result in substrate preference changes in enzyme assays. Therefore, further studies are required to phylogenetically pinpoint the timing and structural basis for the shift towards cinnamic acid methylation preference and to deepen our understanding of molecular evolution of the SABATH enzyme family.