Emergence of isochorismate-based salicylic acid biosynthesis within Brassicales.

Salicylic acid (SA) is a major defense phytohormone. In , the isochorismate (IC) pathway is the primary route for pathogen-induced SA biosynthesis. First, the IC synthase (ICS) catalyzes the isomerization of chorismate to IC in chloroplasts. Second, the chloroplast-localized MATE transporter EDS5 appears to transport IC from chloroplasts to the cytosol. Cytosolic IC is then further converted to SA via the GH3 amino acid-conjugating enzyme PBS3. While this pathway is genetically well-characterized in , its evolutionary origin and conservation remain controversial. In this study, through comprehensive phylogenetic, structural, and functional analyses, we demonstrate that the IC pathway emerged within the Brassicales order in a time span between the divergence of and . The evolution of the IC pathway was driven by three key adaptations during the time span: 1) enhancement of ICS activity, 2) neofunctionalization of after duplication of its ancestral gene, and 3) evolution of a , whose activity is specialized for glutamate-conjugation to IC. Structural modeling and functional assays reveal that an enhanced salt bridge network in ICS enhanced its activity. One of the duplicated genes, EDS5, acquired key amino acid substitutions in the C-lobe, which contributed to the neofunctionalization. In addition, the functional clade, including , is restricted to a Brassicales clade. Taken together, this study addresses the evolutionary trajectory of IC-based SA biosynthesis.