Nitric oxide degradation by potato tuber mitochondria: Evidence for the involvement of external NAD(P)H dehydrogenases

AbstractThe mechanisms of nitric oxide (NO) synthesis in plants have been extensively investigated. NO degradation can be just as important as its synthesis in controlling steady-state levels of NO. Here, we examined NO degradation in mitochondria isolated from potato tubers and the contribution of the respiratory chain to this process. NO degradation was faster in mitochondria energized with NAD(P)H than with succinate or malate. Oxygen consumption and the inner membrane potential were transiently inhibited by NO in NAD(P)H-energized mitochondria, in contrast to the persistent inhibition seen with succinate. NO degradation was abolished by anoxia and superoxide dismutase, which suggested that NO was consumed by its reaction with superoxide anion (O2−). Antimycin-A stimulated and myxothiazol prevented NO consumption in succinate- and malate-energized mitochondria. Although favored by antimycin-A, NAD(P)H-mediated NO consumption was not abolished by myxothiazol, indicating that an additional site of O2− generation, besides complex III, stimulated NO degradation. Larger amounts of O2− were generated in NAD(P)H- compared to succinate- or malate-energized mitochondria. NAD(P)H-mediated NO degradation and O2− production were stimulated by free Ca2+ concentration. Together, these results indicate that Ca2+-dependent external NAD(P)H dehydrogenases, in addition to complex III, contribute to O2− production that favors NO degradation in potato tuber mitochondria

Study of nitric homeostasis in mitochondrial bioenergetics and in plant defense response to pahogen attack

Orientador: Ione SalgadoTese (doutorado) - Universidade Estadual de Campoinas, Instituto de BiologiaResumo: O controle da homeostase do óxido nítrico (NO), determinada pelo balanço entre os processos de síntese e degradação, é essencial para suas funções sinalizadoras. O presente estudo teve como objetivo geral buscar um melhor entendimento da homeostase do NO em plantas, enfocando a importância dessa molécula na bioenergética mitocondrial e na resposta de defesa vegetal ao ataque de patógenos. Inicialmente, foi realizada a caracterização de uma atividade de degradação de NO por mitocôndrias isoladas de tubérculos de batata, observando-se um consumo mitocondrial dependente de sua reação com o ânion superóxido, estimulado na presença de NAD(P)H. Ensaios com diferentes substratos e inibidores respiratórios evidenciaram as NAD(P)H desidrogenases externas, além do complexo III, como os principais sítios de geração de ânion superóxido para o consumo de NO. Por outro lado, numa análise comparativa com mitocôndrias isoladas de fígado de rato, uma atividade NAD(P)H oxidase mitocondrial não associada à cadeia respiratória foi detectada como uma fonte de superóxido, em adição ao vazamento de elétrons nos complexos I e III, para o consumo de NO. Em ambos os casos, a existência de mecanismos mitocondriais de degradação de NO mostrou-se importante para o controle de seus efeitos inibitórios sobre a atividade respiratória. Adicionalmente, a importância da síntese de NO para a defesa vegetal foi analisada utilizando-se como modelo a interação Arabidopsis thaliana-Pseudomonas syringae. Trabalhos anteriores já haviam demonstrado que o mutante de A. thaliana duplo-deficiente para a nitrato redutase (nia1 nia2) apresenta reduzida produção de NO e susceptibilidade à P. syringae, o que poderia resultar de sua prejudicada assimilação de nitrogênio. No presente estudo, plantas nia1 nia2 foram cultivadas com glutamina ou arginina para aumentar os níveis foliares de aminoácidos. Entretanto, esse mutante continuou a desenvolver uma baixa emissão de NO e permaneceu susceptível à infecção bacteriana, indicando que a susceptibilidade não resulta do reduzido conteúdo de aminoácidos. Por outro lado, a fumigação com baixas concentrações do gás NO de plantas nia1 nia2 com os níveis de aminoácidos recuperados restabeleceu a resposta de resistência. Coerentemente, uma análise do perfil transcriptômico utilizando microarranjos de DNA mostrou que o tratamento com NO induziu diversos genes relacionados à defesa em folhas nia1 nia2 infectadas, como aqueles relacionados às vias de sinalização do ácido salicílico e do cálcio, as proteínas relacionadas à patogênese, a reorganização da parede celular e a síntese de compostos com atividade antimicrobiana. Ainda, essa análise indicou novos genes como potenciais alvos do NO, sugerindo aspectos até então desconhecidos do papel dessa molécula sinalizadora na interação fitopatogênica e na fisiologia vegetal. Em especial, destacou-se o possível envolvimento do NO na alteração de transcritos relacionados à sinalização hormonal de forma a permitir um controle atenuador de mecanismos da resposta de defesa.Abstract: The control of nitric oxide (NO) homeostasis, determined by a balance between the rate of synthesis and degradation, is essential for its signaling functions. The present study aimed a better understanding of NO homeostasis in plants, focusing on the importance of this molecule in mitochondrial bioenergetics and in plant defense response to pathogen attack. Initially, we carried out a characterization of an NO degradation activity by mitochondria isolated from potato tubers, observing a superoxide-dependent NO consumption, that was stimulated in the presence of NAD(P)H. Assays with different respiratory substrates and inhibitors evidenced the external NAD(P)H dehydrogenases, in addition to complex III, as the main sites of superoxide anion generation for NO consumption. On the other hand, in a comparative analysis with mitochondria isolated from rat liver, a mitochondrial NAD(P)H oxidase activity, non-associated to the respiratory chain, emerged as a superoxide source, in addition to the electron leakage from complexes I and III, for NO consumption. In both cases, the existence of mitochondrial mechanisms of NO degradation was important for the control of its inhibitory effects on respiratory activity. Additionally, the importance of NO synthesis for plant defense was analyzed using the interaction Arabidopsis thaliana- Pseudomonas syringae as a model. Previous works have shown that the nitrate reductase double-deficient mutant of A. thaliana (nia1 nia2) presents reduced NO production and susceptibility to P. syringae, that could result from its impaired nitrogen assimilation. Here, nia1 nia2 plants were cultivated with glutamine or arginine to increase the leaf amino acid content. Despite this, this mutant continued to develop a low NO emission and remained susceptible to bacterial infection, indicating that the susceptibility does not result from reduced amino acid levels. On the other hand, the fumigation of amino acid-recovered nia1 nia2 plants with low concentrations of NO gas reestablished the resistance response. Accordingly, a transcriptomic analysis using DNA microarrays showed that NO treatment induced diverse defense-related genes in infected nia1 nia2 leaves, as those associated to salicylic acid and calcium signaling pathways, pathogenesis-related proteins, cell wall reorganization and synthesis of antimicrobial compounds. Additionally, this analysis indicated new genes as potential targets of NO action, suggesting previously unknown aspects about the role of this signaling molecule in phytopathogenic interactions. In special, we can highlight the possible involvement of NO in the modulation of transcripts related to hormonal signaling in order to allow an attenuating control of certain mechanisms of the defense response.DoutoradoBioquimicaDoutor em Biologia Funcional e Molecula

Nanoencapsulation Enhances The Post-emergence Herbicidal Activity Of Atrazine Against Mustard Plants

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Poly(epsilon-caprolactone) (PCL) nanocapsules have been recently developed as a modified release system for atrazine, an herbicide that can have harmful effects in the environment. Here, the post-emergence herbicidal activity of PCL nanocapsules containing atrazine was evaluated using mustard (Brassica juncea) as target plant species model. Characterization of atrazine-loaded PCL nanocapsules by nanoparticle tracking analysis indicated a concentration of 7.5 x 10(12) particles mL(-1) and an average size distribution of 240.7 nm. The treatment of mustard plants with nanocapsules carrying atrazine at 1 mg mL(-1) resulted in a decrease of net photosynthesis and PSII maximum quantum yield, and an increase of leaf lipid peroxidation, leading to shoot growth inhibition and the development of severe symptoms. Time course analysis until 72 h after treatments showed that nanoencapsulation of atrazine enhanced the herbicidal activity in comparison with a commercial atrazine formulation. In contrast to the commercial formulation, ten-fold dilution of the atrazine-containing nanocapsules did not compromise the herbicidal activity. No effects were observed when plants were treated with nanocapsules without herbicide compared to control leaves sprayed with water. Overall, these results demonstrated that atrazine-containing PCL nanocapsules provide very effective post-emergence herbicidal activity. More importantly, the use of nanoencapsulated atrazine enables the application of lower dosages of the herbicide, without any loss of efficiency, which could provide environmental benefits.107Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação para o Desenvolvimento da UNESP (FUNDUNESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2013/12322-2, 2011/01872-6]CNPq [573949/2008-5

Nanotechnology Potential in Seed Priming for Sustainable Agriculture

International audienceOur agriculture is threatened by climate change and the depletion of resources and biodiversity. A new agriculture revolution is needed in order to increase the production of crops and ensure the quality and safety of food, in a sustainable way. Nanotechnology can contribute to the sustainability of agriculture. Seed nano-priming is an efficient process that can change seed metabolism and signaling pathways, affecting not only germination and seedling establishment but also the entire plant lifecycle. Studies have shown various benefits of using seed nano-priming, such as improved plant growth and development, increased productivity, and a better nutritional quality of food. Nano-priming modulates biochemical pathways and the balance between reactive oxygen species and plant growth hormones, resulting in the promotion of stress and diseases resistance outcoming in the reduction of pesticides and fertilizers. The present review provides an overview of advances in the field, showing the challenges and possibilities concerning the use of nanotechnology in seed nano-priming, as a contribution to sustainable agricultural practices

Nanoencapsulation Enhances The Post-emergence Herbicidal Activity Of Atrazine Against Mustard Plants.

Poly(epsilon-caprolactone) (PCL) nanocapsules have been recently developed as a modified release system for atrazine, an herbicide that can have harmful effects in the environment. Here, the post-emergence herbicidal activity of PCL nanocapsules containing atrazine was evaluated using mustard (Brassica juncea) as target plant species model. Characterization of atrazine-loaded PCL nanocapsules by nanoparticle tracking analysis indicated a concentration of 7.5 x 10(12) particles mL(-1) and an average size distribution of 240.7 nm. The treatment of mustard plants with nanocapsules carrying atrazine at 1 mg mL(-1) resulted in a decrease of net photosynthesis and PSII maximum quantum yield, and an increase of leaf lipid peroxidation, leading to shoot growth inhibition and the development of severe symptoms. Time course analysis until 72 h after treatments showed that nanoencapsulation of atrazine enhanced the herbicidal activity in comparison with a commercial atrazine formulation. In contrast to the commercial formulation, ten-fold dilution of the atrazine-containing nanocapsules did not compromise the herbicidal activity. No effects were observed when plants were treated with nanocapsules without herbicide compared to control leaves sprayed with water. Overall, these results demonstrated that atrazine-containing PCL nanocapsules provide very effective post-emergence herbicidal activity. More importantly, the use of nanoencapsulated atrazine enables the application of lower dosages of the herbicide, without any loss of efficiency, which could provide environmental benefits

Phytotoxicity evaluation of poly (ɛ-caprolactone) nanocapsules prepared using different methods and compositions in Brassica juncea seeds

abstract: The objective of this study was to analyze the phytotoxic effects of poly (ɛ-caprolactone) (PCL) nanocapsules on mustard (Brassica juncea) seeds. The nanoformulations were prepared using two protocols: PCL nanocapsules containing Tween as a stabilizer (NC TW) and PCL nanocapsules containing poly(vinyl alcohol) (PVA), a polymer that has emulsifying properties (NC PVA). Two experimental systems were used (nanoformulations sprayed on the substrate and as seed treatment), each one in a factorial scheme with two formulations (NC TW and NC PVA) and five dilutions (0%, 25%, 50%, 75%, and 100% of the stock formulation). In general, intermediary concentrations of NC TW showed higher phytotoxicity than NC PVA, as indicated by sharper reductions in germination and increases in the percentage of abnormal seedlings. However, in the substrate treatment, NC PVA (100%) led to a sharper reduction in germination than NC TW in the same dilution. NC TW-treated seeds presented greater water absorption. In an additional assay with nanocapsules labeled with a fluorescent probe (rhodamine B), NC PVA-treated seeds showed higher fluorescence intensity in the seed coat. In the case of NC TW-treated seeds, the fluorescence signal tended to move to the seed interior as the nanocapsule levels increased. Moreover, the seed treatment with this formulation induced sharper increases in water uptake by the seeds than NC PVA. The different effects induced by NC TW and NC PVA could be related to the different characteristics of the nanocapsules, as the lower size and more negative charge of NC TW might have favored their entrance into the seeds

Effect of Nitrogen Addition to the Soil on Atlantic Forest Tree Seedlings

The input of large amounts of fertilizers in agricultural areas may result in nitrogen (N) leakage to nearby forest fragments, which can impact the physiology and growth of trees. The current study aimed to assess the effects of soil N addition on seedlings of four tree species in the Brazilian Atlantic Forest: Croton floribundus and Astronium graveolens (pioneer), Guarea kunthiana and Aspidosperma polyneuron (non-pioneer). The experiment was carried out in a greenhouse condition with three treatments: reference (soil without addition of nutrients), N addition (N: soil with addition of ammoniacal-N), and complete (C: soil with addition of ammoniacal-N and other macro and micronutrients). Croton floribundus seedlings presented higher shoot growth with N addition, mainly in treatment C, and only this treatment increased net photosynthesis. There was great variation in the metabolic responses induced by treatments N and C, with accumulation of nitrate in the leaves and xylem sap only in seedlings in treatment N. In A. graveolens, there was a decrease in transpiration in response to treatments N and C. However, water use efficiency, leaf area, and dry mass increased only in seedlings subjected to treatment C. Regarding metabolic parameters, A. graveolens was little responsive to the treatments. In G. kunthiana seedlings, the treatments decreased net photosynthesis and increased leaf total N. Only treatment N led to decreased stem dry mass and increased nitrate contents in leaves and xylem sap. Aspidosperma polyneuron exhibited no change in growth, but there was an accumulation of N compounds in the leaves for both treatments N and C, which suggests that this species could be a good bioindicator of N addition to the soil. Although influencing different parameters, the results indicate that soil N addition affects the performance of both pioneer and non-pioneer species. Finally, the implications of these results for biomonitoring of N availability in the soil of forest fragments are discussed