Biosynthesis of a major plant immunity hormone, salicylate, changed drastically during evolution of flowering plants

Salicylate (SA) is a key plant immunity hormone, which induces immunity against biotrophic pathogens. Two SA biosynthetic pathways are known: the isochorismate synthase (ICS) and the phenylalanine ammonia-lyase (PAL) pathways. However, the primary pathways used are known for only a few plant species. For example, the ICS pathway evolved within the Brassicales order, and Arabidopsis, a member of this order, primarily uses it. It is challenging to determine the proportions of these pathway contributions using genetic perturbations because the SA biosynthetic pathways are typically regulated by feedback from SA. We developed a simple method to directly measure the relative contributions of these two pathways to SA synthesis using [13C6]glucose feeding to leaves and LC-MS/MS analysis of the leaf extracts. A survey of 16 diverse eudicot species and a monocot species, rice, showed that all these species, except for the Brassicales species with the ICS pathway, mainly use the PAL pathway. The Brassicales species mostly used the ICS pathway but did not use it exclusively: for example, the contribution of the ICS pathway in Arabidopsis was 84% on average. We found that the main pathway in two basal angiosperm species was neither the ICS nor the PAL pathway. Instead, the [13C]-labeling patterns of SA in these species were consistent with possible involvement of a type III polyketide synthase. Thus, during evolution of flowering plants, the main SA biosynthetic pathways changed drastically at least twice. Pathogen effectors targeting SA biosynthetic pathway components likely exerted selection pressure driving these drastic changes.