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[Objective] Salicylic acid(SA) is a phenolic hormone widely distributed in plant tissues, exerting critical regulatory roles in growth, development, and stress resistance. It also serves as a precursor to the pharmaceutical aspirin. SA has attracted increasing attention recently as a key secondary metabolite useful in plant biology and biomedical research. The isochorismate synthase(ICS) pathway has traditionally been considered the conserved route for SA biosynthesis, starting from chorismic acid through the sequential action of ICS1, EDS5, PBS3, and EPS1. However, this view has been challenged by the recently identified phenylalanine ammonia lyase(PAL) pathway, which begins with phenylalanine, proceeds through multiple enzyme-catalyzed reactions to form benzoyl-CoA. Then, the newly discovered enzymes BEBT/OSD2, BBO/BBH/OSD3, and BSH/BSE/OSD4 ultimately synthesize SA. Accumulating evidence suggests the PAL pathway as the ancient and conserved route for SA biosynthesis. A robust extraction and analytical method was developed to accurately detect the intermediates of the two pathways and identify the active one in plants. [Methods] Leaves were flash-frozen in liquid nitrogen, ground into a fine powder using a tissue grinder, and extracted with 80% methanol at a ratio of 1 mL per 0.1 g of the fresh sample. The mixture was sonicated for 10 min and centrifuged at 14,000 g and 4 ℃ for 10 min. The supernatant was transferred to a fresh centrifuge tube. The pellet was re-extracted with 0.5 mL of 100% methanol, and the supernatants from the two extractions were combined. The pooled extract was passed through a 0.22 μm microporous membrane filter and transferred into a brown autosampler vial for metabolic analysis. The major intermediate metabolites of the benzoyl-CoA-dependent PAL pathway in Nicotiana benthamiana—including benzyl benzoate, benzyl salicylate, and SA—were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS). A C18 column was employed for separation and purification. The MS conditions included electrospray ionization, multiple reaction monitoring, and detection in positive and negative ion modes. Standard metabolite solutions were serially diluted to final concentrations of 1, 5, 50, 100, and 500 ng/mL and quantified using calibration curves. [Results] This method exhibited an excellent linearity for the intermediates of SA biosynthesis pathways, with correlation coefficients reaching 1.0, indicating robust sensitivity and accuracy. Arabidopsis thaliana and N. benthamiana leaves were inoculated with Pseudomonas syringae pv. tomato DC3000. In A. thaliana, the levels of the ICS pathway intermediates, isochorismic acid and isochorismic acid-glutamine, were elevated significantly, along with a marked accumulation of SA. In contrast, in N. benthamiana, the levels of these intermediates did not change markedly, whereas SA levels were robustly elevated. Leaves of the N. benthamiana mutants, bbo and bsh, inoculated with DC3000, demonstrated distinct metabolic alterations: benzyl benzoate and benzyl salicylate accumulated in bbo and bsh, respectively. However, in both, the SA content was remarkably reduced compared with the wild type. These results confirm that the PAL pathway is the predominant route for SA biosynthesis in N. benthamiana. [Conclusions] A robust and sensitive UPLC-MS/MS method was established for detecting the key intermediates of the ICS and PAL pathways. It features a straightforward sample pretreatment method, a short analysis time, high detection sensitivity, and broad applicability. This approach not only facilitates the accurate quantification of intermediates but also provides a valuable tool for dissecting pathway dynamics and for advancing the research into SA biosynthesis regulation.
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Basic Information:
DOI：10.16791/j.cnki.sjg.2026.01.017
China Classification Code:Q946;O657.63
Citation Information:
[1]HUANG Yulin,BAI Feng,ZHANG Yang ,et al.Intermediates of the novel phenylalanine ammonia lyase biosynthesis pathway for salicylic acid determined by ultra-performance liquid chromatography-tandem mass spectrometry[J].Experimental Technology and Management,2026,43(01):138-144.DOI:10.16791/j.cnki.sjg.2026.01.017.
Fund Information:
四川大学“人工智能赋能创新型实验技术研究项目”（SCU241053）;四川大学“人工智能赋能创新型实践教育综合改革专项”（2024年度,编号29）; 四川省科技计划项目（2023NSFSC1991）