Unraveling the central puzzle in salicylic acid biosynthesis from phenylalanine

Salicylic acid (SA), a natural phenolic compound originally identified in willow bark, has been used for over 200 years in humans for its anti-inflammatory and analgesic properties. In addition to its medical significance, SA has emerged as a pivotal plant defense hormone over the past three decades, mediating immunity against biotrophic and semibiotrophic pathogens. It plays crucial roles in basal defense, amplification of local immune responses, and establishment of systemic acquired resistance (Peng et al., 2021). In plants, SA biosynthesis was historically thought to originate exclusively from chorismate. Subsequent research revealed two distinct biosynthetic routes: the isochorismate synthase (ICS) pathway and the phenylalanine ammonia lyase (PAL) pathway (Wildermuth et al., 2001). The ICS pathway has been fully established in the dicotyledonous model plant Arabidopsis thaliana. In this pathway, chorismate is converted to isochorismate by ICS1 in plastids, followed by transport to the cytosol via the MATE transporter EDS5. The amidotransferase PBS3 conjugates isochorismate with glutamate to form isochorismate-9-glutamate (IC-9-Glu), a key SA biosynthetic intermediate. IC-9-Glu can either spontaneously decompose into SA or be converted by EPS1 (Rekhter et al., 2019; Torrens-Spence et al., 2019) (Figure 1). Schematic representation of the complete pathways for salicylic acid biosynthesis in plants The enzymes and their abbreviations are as follows: PAL, phenylalanine ammonia lyase; ICS, isochorismate synthase; EDS5, enhanced disease susceptibility 5; PBS3, avrPphB susceptible 3; EPS1: enhanced pseudomonas susceptibility 1; OSD1/CNL, cinnamoyl-CoA ligase; AIM1/CHD, cinnamoyl-CoA hydrogenase-dehydrogenase; KAT, 2,3-ketoacyl-CoA thiolase; OSD2/BEBT, benzoyl-CoA: benzylalcohol benzoyl transferase; OSD3/BB2H/BBH/BBO, benzyl benzoate 2-hydroxylase; OSD4/BSE/BSH, benzyl salicylate hydrolase. Solid arrows represent conversion steps, while dashed arrows indicate biosynthesis routes involving unidentified enzymes, and stacked arrows signify multiple enzymatic steps. Question marks denote unknown biosynthesis pathways. The ICS pathway is highlighted by pale orange and the PAL pathway is highlighted by lavender. The PAL pathway has long been implicated in SA biosynthesis across numerous plant species; however, the metabolic roadmap remains incomplete. Recently, three independent research groups reported groundbreaking studies in Nature, collectively resolving this key pathway for the first time in seed plants. The landmark studies, “Three-step biosynthesis of salicylic acid from benzoyl-CoA in plants” (Liu et al., 2025) by Yuelin Zhang's team at Sichuan University, “Complete biosynthesis of salicylic acid from phenylalanine in plants” (Zhu et al., 2025) from Kewei Zhang's team at Zhejiang Normal University, and “Deciphering phenylalanine-derived salicylic acid biosynthesis in plants” (Wang et al., 2025a) by Ronghui Pan's team at Zhejiang University, all reveal a conserved PAL–SA pathway involving benzoyl-CoA biosynthesis with benzyl benzoate (BB) as a critical intermediate in plants (Figure 1). These discoveries fundamentally challenge the long-standing hypothesis by demonstrating that the pivotal hydroxylation step in SA biosynthesis occurs at BB rather than benzoic acid (BA). In these studies, researchers employed analogous high-throughput forward genetic screening strategies and co-expression analysis to identify core genes involved in SA biosynthesis. In tobacco, an ethyl methane sulfonate (EMS)-mutagenized population was screened to isolate mutants displaying reduced SA accumulation following Pst DC3000 infection. NbBEBT was identified using mapping-by-sequencing and clustered regularly interspaced small palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene-edited lines, with subsequent co-expression analyses of pathogen-induced expression patterns uncovering NbBBO and NbBSH as additional contributors to SA biosynthetic pathways (Liu et al., 2025). In rice, OSD1 was identified via map-based cloning from an EMS-mutagenized library, followed by co-expression analysis using OSD1 as a bait to screen for OSD2, OSD3, and OSD4 (Zhu et al., 2025). Alternatively, candidate genes in rice can be screened by performing co-expression analysis using OsCNL1, the most functionally specific enzyme among the known enzymes of the PAL pathway (Wang et al., 2025b), as the bait to identify BEBT, BBH, and BSE (Wang et al., 2025a). The biosynthetic logic was elucidated through an integrated approach combining in vitro enzymatic activity assays, kinetic analyses, subcellular localization assays, substrate/intermediate feeding experiments, and isotope-labeling tracer analyses. To sum up, the discovery of a conserved PAL-dependent pathway for SA biosynthesis in seed plants originates with the conversion of phenylalanine to trans-cinnamic acid by PAL. Trans-cinnamic acid is converted to benzoyl-CoA via the peroxisomal β-oxidation pathway, which involves three key enzymes: cinnamate-CoA ligase (CNL/OSD1) activates trans-cinnamic acid to cinnamoyl-CoA (Wang et al., 2024), followed by its sequential conversion to benzoyl-CoA through the actions of cinnamoyl-CoA hydratase/dehydrogenase (CHD/AIM1) and 3-ketoacyl-CoA thiolase (KAT). The resultant benzoyl-CoA is conjugated with benzyl alcohol by the peroxisomal benzoyl-CoA: benzyl alcohol benzoyltransferase (BEBT/OSD2), forming BB. Subsequently, BB is hydroxylated to benzyl salicylate (BS) by the endoplasmic reticulum (ER) membrane-resident BB hydroxylase/oxidase (BBH/BBO/OSD3). Finally, BS is hydrolyzed to SA by the cytoplasmic carboxylesterase benzyl salicylate esterase/hydrolase (BSE/BSH/OSD4) (Figure 1). In addition to deciphering the core PAL–SA biosynthetic machinery in plants, they also conducted comparative genomic analyses across diverse plant species to reconstruct its evolutionary trajectory. Systematic investigations across 25 species representing 11 major plant lineages (from Rhodophyta to Eudicots) (Zhu et al., 2025), complemented by conservation analyses of key gene homologs in 70 seed plants (Liu et al., 2025), reveal a modular, stepwise assembly of the PAL–SA pathway. The ancestral β-oxidation complex (CNL-AIM1-KAT) responsible for benzoate derivative synthesis first emerged in Chlorophyta, with AIM1 orthologs predating this radiation in Rhodophytes (Jia et al., 2023). Notably, CNL was subsequently lost during early land plant evolution (Zhu et al., 2025). The pathway subsequently acquired: (i) the cytochrome P450 hydroxylase BBH/BBO/OSD3 in basal vascular plants, which became widely conserved in tracheophytes, and (ii) the ester metabolic components BEBT/OSD2 and BSE/BSH/OSD4 coincident with gymnosperm emergence (Zhu et al., 2025). This indicates pathway maturation prior to gymnosperm divergence, though Arabidopsis underwent specific losses of BBH/BBO/OSD3 and BSE/BSH/OSD4 (Liu et al., 2025). Collectively, these findings demonstrate that a fully functional PAL–SA pathway was not complete until the evolutionary emergence of gymnosperms, with this stepwise-assembled pathway being broadly conserved across seed plants—with the notable exception of Arabidopsis. These findings also redefine the spatial and biochemical organization of SA biosynthesis, resolving a decades-old mystery in plant metabolism. Historically, SA production was hypothesized to occur via BA hydroxylation catalyzed by a putative soluble P450 enzyme (BA2H)—a presumption maintained for over 30 years despite the enzyme's molecular identity remaining elusive. These studies conclusively demonstrate an alternative pathway operating across multiple subcellular compartments: BB is synthesized in peroxisomes, translocated to the cytosol, and then hydroxylated by ER membrane-bound BBH/BBO/OSD3 to form BS, and cytosolic hydrolysis of BS to SA (Zhu et al., 2025; Wang et al., 2025a). The compartmentalized nature of this pathway provides critical insights into SA regulation. Notably, rice's exceptional capacity to accumulate high SA levels suggests additional spatial control mechanisms, potentially involving subcellular sequestration in specialized organelles or biomolecular condensates to mitigate cytotoxicity. Overall, the identification of BBH/BBO/OSD3 as the authentic P450 enzyme fulfilling the long-postulated hydroxylation function resolves a major gap in our understanding of SA biosynthesis, while the multi-compartment pathway architecture explains how plants achieve both high production capacity and safe storage of this phytohormone. We appreciate support from the Project of Sanya Yazhou Bay Science and Technology City (SCKJ-JYRC-2023-18), the Natural Science Foundation of Hainan Province (323RC423), the Hainan University Startup Fund (RZ2100003223), and the High-performance Computing Platform of YZBSTCACC. We sincerely apologize to colleagues whose work could not be cited or discussed due to space limitations. The authors declare no conflicts of interest. Y.L. and J.L. conceived the commentary. Y.L. wrote the draft and J.L. revised the manuscript. All authors read and approved the final manuscript.