Abstract Salicylic acid (SA) biosynthesis in plants occurs via the isochorismate synthase (ICS) and phenylalanine ammonia-lyase (PAL) pathways. The critical steps from benzyl-CoA to SA in the PAL-mediated pathway remain unknown. To probe benzenoid metabolism, we generated CRISPR/Cas9-mediated knockouts of benzaldehyde synthase in poplar. These plants produce less benzyl benzoate, benzyl salicylate and SA, yet accumulate more benzoic acid. We show that HSR203J encodes a carboxylesterase that specifically hydrolyzes benzyl salicylate. Virus-induced gene silencing (VIGS) of HSR203J in Nicotiana benthamiana led to reduced benzyl salicylate hydrolysis to SA. Based on these data, we propose a biosynthesis model and provide evidence that benzoyl-CoA is esterified to benzyl benzoate and converted to benzyl salicylate, which then releases SA. In addition, we identified a pathogen-induced cytochrome P450 encoded by HSR515 as a putative benzyl benzoate 2-hydroxylase. VIGS-mediated suppression of HSR515 in N. benthamiana reduced the conversion of benzyl benzoate to SA. Phylogenetic analyses indicated that Brassicaceae genomes do not contain HSR203J and HSR515 orthologs, whereas these genes are present in other vascular plants. These findings represent an important advance in our understanding of SA biosynthesis and identify missing steps in the PAL-mediated SA biosynthetic pathway.
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