Salicylic acid biosynthesis is not from phenylalanine in Arabidopsis

The phytohormone salicylic acid (SA) regulates biotic and abiotic stress responses in plants. Two distinct biosynthetic pathways for SA have been well documented in plants: the isochorismate (IC) pathway in the chloroplast and the phenylalanine ammonia-lyase (PAL) pathway in the cytosol. However, there has been no solid evidence that the PAL pathway contributes to SA biosynthesis. Here, we report that feeding Arabidopsis thaliana with Ring-13 C-labeled phenylalanine (13 C6 -Phe) resulted in incorporation of the 13 C label not into SA, but into its isomer 4-hydroxybenzoic acid (4-HBA) instead. We obtained similar results when feeding 13 C6 -Phe to the SA-deficient ics1 ics2 mutant and the SA-hyperaccumulating mutant s3h s5h. Notably, we detected 13 C6 -SA when 13 C6 -benzoic acid (BA) was provided, suggesting that SA can be synthesized from BA. Furthermore, despite the substantial accumulation of SA upon pathogen infection, we did not observe incorporation of 13 C label from Phe into SA. We also did not detect 13 C6 -SA in PAL-overexpressing lines in the kfb01 kfb02 kfb39 kfb50 background after being fed 13 C6 -Phe, although endogenous PAL levels were dramatically increased. Based on these combined results, we propose that SA biosynthesis is not from Phe in Arabidopsis. These results have important implications for our understanding of the SA biosynthetic pathway in land plants.