RESUMEN
Acibenzolar-S-methyl (ASM) is the most commercially successful biological antibacterial agent used for crop through systemic acquired resistance (SAR). In this study, a reproducible and accurate procedure, based on the spectrophotometric/microplate reader analysis, has been developed to detect ASM in tobacco leaves. This method involves oxidation of methyl mercaptan by the Ellman's reagent 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB) to form the yellow derivative 5'-thio-2-nitrobenzoic acid (TNB2-), measurable at 412 nm. Methyl mercaptan can be produced by either the ASM transesterification with methanol or the SA-binding protein 2 (SABP2)-catalyzed ASM hydrolysis. The proportions of methanol, reaction time, temperature, the concentrations of EDTA and DTNB were optimized in a 96-well plate. The calibration curve of ASM was linear over the range of 25.2-315 µg g-1. The results of the intra- and inter-day accuracy and precision data were within the FDA acceptance criteria. With ASM as substrate, the turnover number of SABP2 was determined, with the kcat value of 31.1 min-1 using the Michaelis-Menten equation. In tobacco plants treated with 100 µM ASM, it was decreased as time elapsed in treated tobacco, reaching negligible values 72 h after treatment. The optimized method was applied for the determination of ASM transesterification with methanol and the kinetic data determination of SABP2-catalyzed ASM hydrolysis.
Asunto(s)
Metanol , Nicotiana , Proteínas Portadoras/metabolismo , Catálisis , Ácido Ditionitrobenzoico/metabolismo , Hidrólisis , Compuestos de Sulfhidrilo/análisis , TiadiazolesRESUMEN
An alpha/ beta hydrolase annotated as a putative salicylate esterase within the genome of a species of Paenibacillus previously identified from differential and selective growth on Kraft lignin was structurally and functionally characterised. Feruloyl esterases are key to the degradation of lignin in several bacterial species and although this activity was investigated, no such activity was observed. The crystal structure of the Paenibacillus esterase, here denoted as PnbE, was determined at 1.32 Å resolution, showing high similarity to Nicotiana tabacum salicylic acid binding protein 2 from the protein database. Structural similarities between these two structures across the core domains and key catalytic residues were observed, with superposition of catalytic residues giving an RMSD of 0.5 Å across equivalent Cα atoms. Conversely, the cap domains of PnbE and Nicotiana tabacum SABP2 showed greater divergence with decreased flexibility in the PnbE cap structure. Activity of PnbE as a putative methyl salicylate esterase was supported with binding studies showing affinity for salicylic acid and functional studies showing methyl salicylate esterase activity. We hypothesise that this activity could enrich Paenibacillus sp. within the rhizosphere by increasing salicylic acid concentrations within the soil.
Asunto(s)
Hidrolasas/metabolismo , Nicotiana/enzimología , Nicotiana/metabolismo , Paenibacillus/enzimología , Paenibacillus/metabolismo , Hidrolasas/genética , Paenibacillus/genética , Rizosfera , Ácido Salicílico/metabolismo , Nicotiana/genéticaRESUMEN
Salicylic acid (SA) is a phytohormone that plays important roles in many aspects of plant life, notably in plant defenses against pathogens. Key mechanisms of SA signal transduction pathways have now been uncovered. Even though details are still missing, we understand how SA production is regulated and which molecular machinery is implicated in the control of downstream transcriptional responses. The NPR1 pathway has been described to play the main role in SA transduction. However, the mode of SA perception is unclear. NPR1 protein has been shown to bind SA. Nevertheless, NPR1 action requires upstream regulatory events (such as a change in cell redox status). Besides, a number of SA-induced responses are independent from NPR1. This shows that there is more than one way for plants to perceive SA. Indeed, multiple SA-binding proteins of contrasting structures and functions have now been identified. Yet, all of these proteins can be considered as candidate SA receptors and might have a role in multinodal (decentralized) SA input. This phenomenon is unprecedented for other plant hormones and is a point of discussion of this review.
Asunto(s)
Redes y Vías Metabólicas , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Plantas/metabolismo , Ácido Salicílico/metabolismo , Transducción de Señal , Enfermedades de las Plantas , Reguladores del Crecimiento de las Plantas/química , Proteínas de Plantas/química , Ácido Salicílico/química , Estrés Fisiológico , Relación Estructura-ActividadRESUMEN
Salicylic acid (SA) plays a critical role in plant defense against pathogen invasion. SA-induced viral defense in plants is distinct from the pathways mediating bacterial and fungal defense and involves a specific pathway mediated by mitochondria; however, the underlying mechanisms remain largely unknown. The SA-binding activity of the recombinant tomato (Solanum lycopersicum) alpha-ketoglutarate dehydrogenase (Slα-kGDH) E2 subunit of the tricarboxylic acid (TCA) cycle was characterized. The biological role of this binding in plant defenses against tobacco mosaic virus (TMV) was further investigated via Slα-kGDH E2 silencing and transient overexpression in plants. Slα-kGDH E2 was found to bind SA in two independent assays. SA treatment, as well as Slα-kGDH E2 silencing, increased resistance to TMV. SA did not further enhance TMV defense in Slα-kGDH E2-silenced tomato plants but did reduce TMV susceptibility in Nicotiana benthamiana plants transiently overexpressing Slα-kGDH E2. Furthermore, Slα-kGDH E2-silencing-induced TMV resistance was fully blocked by bongkrekic acid application and alternative oxidase 1a silencing. These results indicated that binding by Slα-kGDH E2 of SA acts upstream of and affects the mitochondrial electron transport chain, which plays an important role in basal defense against TMV. The findings of this study help to elucidate the mechanisms of SA-induced viral defense.
Asunto(s)
Complejo Cetoglutarato Deshidrogenasa/metabolismo , Mitocondrias/metabolismo , Fosforilación Oxidativa , Ácido Salicílico/metabolismo , Solanum lycopersicum/inmunología , Solanum lycopersicum/virología , Virus del Mosaico del Tabaco/fisiología , Respiración de la Célula , Resistencia a la Enfermedad/inmunología , Transporte de Electrón , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Solanum lycopersicum/enzimología , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/virología , Proteínas de Plantas/metabolismo , Nicotiana/inmunologíaRESUMEN
Salicylic acid (SA) is a small phenolic molecule that not only is the active ingredient in the multi-functional drug aspirin, but also serves as a plant hormone that affects diverse processes during growth, development, responses to abiotic stresses and disease resistance. Although a number of SA-binding proteins (SABPs) have been identified, the underlying mechanisms of action of SA remain largely unknown. Efforts to identify additional SA targets, and thereby elucidate the complex SA signaling network in plants, have been hindered by the lack of effective approaches. Here, we report two sensitive approaches that utilize SA analogs in conjunction with either a photoaffinity labeling technique or surface plasmon resonance-based technology to identify and evaluate candidate SABPs from Arabidopsis. Using these approaches, multiple proteins, including the E2 subunit of α-ketoglutarate dehydrogenase and the glutathione S-transferases GSTF2, GSTF8, GSTF10 and GSTF11, were identified as SABPs. Their association with SA was further substantiated by the ability of SA to inhibit their enzymatic activity. The photoaffinity labeling and surface plasmon resonance-based approaches appear to be more sensitive than the traditional approach for identifying plant SABPs using size-exclusion chromatography with radiolabeled SA, as these proteins exhibited little to no SA-binding activity in such an assay. The development of these approaches therefore complements conventional techniques and helps dissect the SA signaling network in plants, and may also help elucidate the mechanisms through which SA acts as a multi-functional drug in mammalian systems.