Evolution and diversification of PAL-mediated salicylic acid biosynthesis in Rosaceae

Abstract Salicylic acid (SA) is a central phytohormone in plant immunity and stress responses, yet the evolutionary dynamics of its phenylalanine ammonia-lyase (PAL)-mediated biosynthetic route remain poorly understood despite recent biochemical advances. As the original source of SA, Spiraea (Rosaceae) holds historical and evolutionary significance for studying SA biosynthesis. Here, we generated a chromosome-level genome assembly of Spiraea chinensis and integrated comparative genomics, transcriptomic, and targeted metabolite profiling to investigate the evolutionary diversification of SA biosynthesis across Rosaceae. Phylogenomics places S. chinensis in the subfamily Amygdaloideae, diverging from other genera ~57.8 Mya. Extensive chromosome fission–fusion events and lineage-specific whole-genome duplication (WGD) have driven karyotype diversification across Rosaceae. Comparative analyses revealed the PAL-mediated route as the dominant SA biosynthetic pathway across Rosaceae, with WGD-driven expansion in Amygdaloideae and combined WGD- and small-scale duplication (SSD)-derived origins in Rosoideae. WGD-derived PAL-route genes largely retained synteny and stable high expression, whereas lineage-specific SSD-derived paralogs exhibited reduced synteny and variable expression, consistent with post-duplication regulatory divergence and subfunctionalization. Transcriptome analyses revealed pronounced tissue-specific expression of PAL-route genes across Rosaceae, and UPLC–MS/MS profiling further demonstrated differential SA accumulation in S. chinensis, with the highest levels in branches (606–1038 ng/g FW), followed by leaves (183–432 ng/g FW) and flowers (42–56 ng/g FW), supporting active SA biosynthesis in both vegetative and reproductive tissues. Collectively, our results establish the PAL-mediated pathway as the primary and evolutionarily conserved route of SA biosynthesis in Rosaceae and demonstrate how genome dynamics and regulatory diversification jointly drive the evolutionary innovation within this pathway.