Person: Buezo Bravo, Javier
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Buezo Bravo
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Javier
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Ciencias
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IMAB. Research Institute for Multidisciplinary Applied Biology
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0000-0002-6287-1587
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811160
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Publication Open Access IAOx induces the SUR phenotype and differential signalling from IAA under different types of nitrogen nutrition in Medicago truncatula roots(Elsevier, 2019) Buezo Bravo, Javier; Esteban Terradillos, Raquel; Cornejo Ibergallartu, Alfonso; López Gómez, Pedro; Marino Bilbao, Daniel; Chamizo Ampudia, Alejandro; Gil Idoate, María José; Martínez Merino, Víctor; Morán Juez, José Fernando; Zientziak; Institute for Multidisciplinary Research in Applied Biology - IMAB; Institute for Advanced Materials and Mathematics - INAMAT2; Ciencias; Universidad Pública de Navarra / Nafarroako Unibertsitate PublikoaIndole-3-acetaldoxime (IAOx) is a particularly relevant molecule as an intermediate in the pathway for tryptophan-dependent auxin biosynthesis. The role of IAOx in growth-signalling and root phenotype is poorly studied in cruciferous plants and mostly unknown in non-cruciferous plants. We synthesized IAOx and applied it to M. truncatula plants grown axenically with NO3-, NH4+ or urea as the sole nitrogen source. During 14 days of growth, we demonstrated that IAOx induced an increase in the number of lateral roots, especially under NH4+ nutrition, while elongation of the main root was inhibited. This phenotype is similar to the phenotype known as “superroot” previously described in SUR1- and SUR2-defective Arabidopsis mutants. The effect of IAOx, IAA or the combination of both on the root phenotype was different and dependent on the type of N-nutrition. Our results also showed the endogenous importance of IAOx in a legume plant in relation to IAA metabolism, and suggested IAOx long-distance transport depending on the nitrogen source provided. Finally, our results point out to CYP71A as the major responsible enzymes for IAA synthesis from IAOx.Publication Open Access A study of the interface of gold nanoparticles conjugated to cowpea fe-superoxide dismutase(MDPI, 2022) Tellechea Malda, Edurne; Asensio, Aarón C.; Ciáurriz Gortari, Paula; Buezo Bravo, Javier; López Gómez, Pedro; Urra Rodríguez, Marina; Morán Juez, José Fernando; Ciencias; Zientziak; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa (Res 309/2022)The iron superoxide dismutase (FeSOD) is a first barrier to defend photosynthetic organisms from superoxide radicals. Although it is broadly present in plants and bacteria, FeSODs are absent in animals. They belong to the same phylogenic family as Mn-containing SODs, which are also highly efficient at detoxifying superoxide radicals. In addition, SODs can react with peroxynitrite, and FeSOD enzyme has already been used to evaluate the anti-nitrative capacity of plant antioxidants. Gold nanoparticles (AuNPs) have been shown to significantly improve the functionality and the efficiency of ligands, providing they are properly assembled. In this work, the characteristics of the recombinant cowpea (Vigna unguiculata) FeSOD (rVuFeSOD) immobilized onto AuNPs were investigated as a function of (1) NP surface chemistry and (2) biofunctionalization methods, either physical adsorption or covalent bonding. The NP surface chemistry was studied by varying the concentration of the ligand molecule 11-mercaptoundecanoic acid (MUA) on the NP surface. The coverage and activity of the protein on AuNPs was determined and correlated to the surface chemistry and the two biofunctionalization methods. rVuFeSOD–AuNPs conjugate stability was monitored through absorption measurements, agarose gel electrophoresis and DLS, enzymatic activity by a colorimetric assay and by in-gel activity assay, and coverage was measured by colorimetric assay. When using physical adsorption, the NP is the most perturbing agent for the activity of the enzyme. In contrast, only the NP coverage was affected by MUA ligand concentration. However, during covalent attachment, both the NP and the concentration of MUA on the surface influenced the enzyme activity, while the coverage of the NP remained constant. The results evidence the importance of the biomolecule and AuNP interaction for the functionality of the hybrid. These strategies can be used to develop electrochemical biosensors for O2•− and for peroxynitrite in biomedical applications.Publication Embargo A new oxidative pathway of nitric oxide production from oximes in plants(Cell Press, 2024) López Gómez, Pedro; Buezo Bravo, Javier; Urra Rodríguez, Marina; Cornejo Ibergallartu, Alfonso; Esteban Terradillos, Raquel; Fernández de los Reyes, Jorge; Urarte Rodríguez, Estíbaliz; Rodríguez-Dobreva, Estefanía; Chamizo Ampudia, Alejandro; Eguaras, Alejandro; Wolf, Sebastian; Marino Bilbao, Daniel; Martínez Merino, Víctor; Morán Juez, José Fernando; Ciencias; Zientziak; Institute for Multidisciplinary Research in Applied Biology - IMAB; Institute for Advanced Materials and Mathematics - INAMAT2Nitric oxide (NO) is an essential reactive oxygen species and a signal molecule in plants. Although several studies have proposed the occurrence of oxidative NO production, only reductive routes for NO production, such as the nitrate (NO-3) -upper-reductase pathway, have been evidenced to date in land plants. However, plants grown axenically with ammonium as the sole source of nitrogen exhibit contents of nitrite and NO3, evidencing the existence of a metabolic pathway for oxidative production of NO. We hypothesized that ox- imes, such as indole-3-acetaldoxime (IAOx), a precursor to indole-3-acetic acid, are intermediate oxidation products in NO synthesis. We detected the production of NO from IAOx and other oximes catalyzed by peroxidase (POD) enzyme using both 4-amino-5-methylamino-20,70-difluorescein fluorescence and chem- iluminescence. Flavins stimulated the reaction, while superoxide dismutase inhibited it. Interestingly, mouse NO synthase can also use IAOx to produce NO at a lower rate than POD. We provided a full mech- anism for POD-dependent NO production from IAOx consistent with the experimental data and supported by density functional theory calculations. We showed that the addition of IAOx to extracts from Medicago truncatula increased the in vitro production of NO, while in vivo supplementation of IAOx and other oximes increased the number of lateral roots, as shown for NO donors, and a more than 10-fold increase in IAOx dehydratase expression. Furthermore, we found that in vivo supplementation of IAOx increased NO pro- duction in Arabidopsis thaliana wild-type plants, while prx33-34 mutant plants, defective in POD33-34, had reduced production. Our data show that the release of NO by IAOx, as well as its auxinic effect, explain the superroot phenotype. Collectively, our study reveals that plants produce NO utilizing diverse molecules such as oximes, POD, and flavins, which are widely distributed in the plant kingdom, thus intro- ducing a long-awaited oxidative pathway to NO production in plants. This knowledge has essential impli- cations for understanding signaling in biological systems.Publication Open Access Tryptophan levels as a marker of auxins and nitric oxide signaling(MDPI, 2022) López Gómez, Pedro; Smith, Edward N.; Bota, Pedro; Cornejo Ibergallartu, Alfonso; Urra Rodríguez, Marina; Buezo Bravo, Javier; Morán Juez, José Fernando; Zientziak; Institute for Advanced Materials and Mathematics - INAMAT2; Institute for Multidisciplinary Research in Applied Biology - IMAB; Ciencias; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate PublikoaThe aromatic amino acid tryptophan is the main precursor for indole-3-acetic acid (IAA), which involves various parallel routes in plants, with indole-3-acetaldoxime (IAOx) being one of the most common intermediates. Auxin signaling is well known to interact with free radical nitric oxide (NO) to perform a more complex effect, including the regulation of root organogenesis and nitrogen nutrition. To fathom the link between IAA and NO, we use a metabolomic approach to analyze the contents of low-molecular-mass molecules in cultured cells of Arabidopsis thaliana after the application of S-nitrosoglutathione (GSNO), an NO donor or IAOx. We separated the crude extracts of the plant cells through ion-exchange columns, and subsequent fractions were analyzed by gas chromatography-mass spectrometry (GC-MS), thus identifying 26 compounds. A principal component analysis (PCA) was performed on N-metabolism-related compounds, as classified by the Kyoto Encyclopedia of Genes and Genomes (KEGG). The differences observed between controls and treatments are mainly explained by the differences in Trp contents, which are much higher in controls. Thus, the Trp is a shared response in both auxin- and NO-mediated signaling, evidencing some common signaling mechanism to both GSNO and IAOx. The differences in the low-molecularmass- identified compounds between GSNO- and IAOx-treated cells are mainly explained by their concentrations in benzenepropanoic acid, which is highly associated with IAA levels, and salicylic acid, which is related to glutathione. These results show that the contents in Trp can be a marker for the study of auxin and NO signaling.Publication Open Access The importance of the urea cycle and its relationships to polyamine metabolism during ammonium stress in Medicago truncatula(Oxford University Press, 2022) Urra Rodríguez, Marina; Buezo Bravo, Javier; Royo Castillejo, Beatriz; Cornejo Ibergallartu, Alfonso; López Gómez, Pedro; Cerdán Ruiz, Daniel; Esteban Terradillos, Raquel; Martínez Merino, Víctor; Gogorcena, Yolanda; Tavladoraki, Paraskevi; Morán Juez, José Fernando; Zientziak; Institute for Advanced Materials and Mathematics - INAMAT2; Institute for Multidisciplinary Research in Applied Biology - IMAB; Ciencias; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa; Gobierno de Navarra / Nafarroako GobernuarenThe ornithine–urea cycle (urea cycle) makes a signifcant contribution to the metabolic responses of lower photosynthetic eukaryotes to episodes of high nitrogen availability. In this study, we compared the role of the plant urea cycle and its relationships to polyamine metabolism in ammonium-fed and nitrate-fed Medicago truncatula plants. High ammonium resulted in the accumulation of ammonium and pathway intermediates, particularly glutamine, arginine, ornithine, and putrescine. Arginine decarboxylase activity was decreased in roots, suggesting that the ornithine decarboxylase-dependent production of putrescine was important in situations of ammonium stress. The activity of copper amine oxidase, which releases ammonium from putrescine, was signifcantly decreased in both shoots and roots. In addition, physiological concentrations of ammonium inhibited copper amine oxidase activity in in vitro assays, supporting the conclusion that high ammonium accumulation favors putrescine synthesis. Moreover, early supplementation of plants with putrescine avoided ammonium toxicity. The levels of transcripts encoding urea-cyclerelated proteins were increased and transcripts involved in polyamine catabolism were decreased under high ammonium concentrations. We conclude that the urea cycle and associated polyamine metabolism function as important protective mechanisms limiting ammonium toxicity in M. truncatula. These fndings demonstrate the relevance of the urea cycle to polyamine metabolism in higher plants.