The phytohormone salicylic acid (SA) plays essential roles in biotic and abiotic responses, plant development, and leaf senescence. 2,5-Dihydroxybenzoic acid (2,5-DHBA or gentisic acid) is one of the most commonly occurring aromatic acids in green plants and is assumed to be generated from SA, but the enzymes involved in its production remain obscure. DMR6 (Downy Mildew Resistant6; At5g24530) has been proven essential in plant immunity of Arabidopsis (Arabidopsis thaliana), but its biochemical properties are not well understood. Here, we report the discovery and functional characterization of DMR6 as a salicylic acid 5-hydroxylase (S5H) that catalyzes the formation of 2,5-DHBA by hydroxylating SA at the C5 position of its phenyl ring in Arabidopsis. S5H/DMR6 specifically converts SA to 2,5-DHBA in vitro and displays higher catalytic efficiency (K_cat/K_m = 4.96 × 10⁴m⁻¹ s⁻¹) than the previously reported S3H (K_cat/K_m = 6.09 × 10³m⁻¹ s⁻¹) for SA. Interestingly, S5H/DMR6 displays a substrate inhibition property that may enable automatic control of its enzyme activities. The s5h mutant and s5hs3h double mutant overaccumulate SA and display phenotypes such as a smaller growth size, early senescence, and a loss of susceptibility to Pseudomonas syringae pv tomato DC3000. S5H/DMR6 is sensitively induced by SA/pathogen treatment and is expressed widely from young seedlings to senescing plants, whereas S3H is more specifically expressed at the mature and senescing stages. Collectively, our results disclose the identity of the enzyme required for 2,5-DHBA formation and reveal a mechanism by which plants fine-tune SA homeostasis by mediating SA 5-hydroxylation.

Perception of SA by its receptors is required for activation of NHP biosynthesis during SAR, plays crucial roles in PTI and ETI, and is involved in regulating S

Salicylic acid (SA) is a pivotal phytohormone for plant responses to biotic and abiotic stresses. Plants have evolved two pathways to produce SA: the isochorismate synthase and phenylalanine ammonia lyase (PAL) pathways¹. Whereas the isochorismate synthase pathway has been fully identified^2–4, the PAL pathway remains incomplete. Here we report the full characterization of the PAL pathway for SA biosynthesis via functional analysis of rice (Oryza sativa) SA-DEFICIENT GENE 1 (OSD1) to OSD4. The cinnamoyl-coenzyme A (CoA) ligase OSD1 catalyses the conversion of trans-cinnamic acid to cinnamoyl-CoA, which is subsequently transformed to benzoyl-CoA via the β-oxidative pathway in peroxisomes. The resulting benzoyl-CoA is further converted to benzyl benzoate by the peroxisomal benzoyltransferase OSD2. Benzyl benzoate is subsequently hydroxylated to benzyl salicylate by the endoplasmic reticulum membrane-resident cytochrome P450 OSD3, which is ultimately hydrolysed to salicylic acid by the cytoplasmic carboxylesterase OSD4. Evolutionary analyses reveal that the PAL pathway was first assembled before the divergence of gymnosperms and has been conserved in most seed plants. Activation of the PAL pathway in rice significantly enhances salicylic acid levels and plant immunity. Completion of the PAL pathway provides critical insights into the primary salicylic acid biosynthetic pathway across plant species and offers a precise target for modulating crop immunity.

The purple color of the ripe Eugenia jambolana fruit is attributed to anthocyanins, a class of plant pigments that has attracted immense attention due to the potential health benefits of these compounds. There is disagreement in the literature on whether E. jambolana fruit anthocyanins are found as 3,5- or 3-diglucosides. Therefore, we used a combination of HPLC-UV, tandem LC-MS, and NMR techniques to identify the structures of anthocyanins present in E. jambolana fruit collected in the USA and India. Our results indicate that the anthocyanins from both locations occur as 3,5-, but not 3-diglucosides, of delphinidin, cyanidin, petunidin, peonidin, and malvidin. This is the first report to use a combination of spectrometric and spectroscopic methods to identify unequivocally the structures of E. jambolana fruit anthocyanins.