S-Nitrosoglutathione is a component of wound- and salicylic acid-induced systemic responses in Arabidopsis thaliana

S-Nitrosoglutathione (GSNO) is a bioactive, stable, and mobile reservoir of nitric oxide (NO), and an important player in defence responses to herbivory and pathogen attack in plants. It has been demonstrated previously that GSNO reductase (GSNOR) is the main enzyme responsible for the in vivo control of intracellular levels of GSNO. In this study, the role of S-nitrosothiols, in particular of GSNO, in systemic defence responses in Arabidopsis thaliana was investigated further. It was shown that GSNO levels increased rapidly and uniformly in injured Arabidopsis leaves, whereas in systemic leaves GSNO was first detected in vascular tissues and later spread over the parenchyma, suggesting that GSNO is involved in the transmission of the wound mobile signal through the vascular tissue. Moreover, GSNO accumulation was required to activate the jasmonic acid (JA)-dependent wound responses, whereas the alternative JA-independent wound-signalling pathway did not involve GSNO. Furthermore, extending previous work on the role of GSNOR in pathogenesis, it was shown that GSNO acts synergistically with salicylic acid in systemic acquired resistance activation. In conclusion, GSNOR appears to be a key regulator of systemic defence responses, in both wounding and pathogenesis

Estudio funcional de la formaldehído deshidrogenasa dependiente de glutatión de Arabidopsis Thaliana : aplicaciones en fitoremediacion /

Consultable des del TDXTítol obtingut de la portada digitalitzadaEl formaldehído es un agente citotóxico que provoca mutaciones in el DNA, ha sido clasificado como un carcinógeno. Se produce durante el metabolismo celular normal, pero su concentración aumenta con el estrés oxidativo. El formaldehído es un polucionante atmosférico (especialmente de interiores) y de aguas residuales. El principal sistema enzimático que metaboliza el formaldehído en los organismos eucariotas es mediante la formaldehído deshidrogenasa dependiente de glutatión. La formaldehído dehidrogenasa dependiente de glutatión, también, llamada ADH clase III, en presencia del NAD+ cataliza la oxidación de formaldehído. El sustrato real del enzima es el S-hidroximetilglutatión que se forma de la reacción espontánea entre el formaldehído y el glutatión. La FALDH se encuentra universalmente distribuida en todos los organismos, tanto en el reino vegetal como animal. Ambas propiedades del enzima, distribución ubicua y alta conservación de secuencia, sugieren que la función principal de este enzima es metabolismo del formaldehído tanto endógeno como exógeno. Se ha estudiado el efecto de sobreexpresión del gen de la FALDH en dos organismos Sacaromyces cerevisiae y Arabidopsis thaliana. La sobre expresión en un vector multicopias del gen FALDH en levadura confiere a ésta la capacidad de resistir a altas concentraciones de formaldehído (2 mM). También, se han obtenido varias plantas transgénicas que sobreexpresan la FALDH de Arabidopsis. El estudio de la capacidad de las diferentes líneas transgénicas de metabolizar concentraciones altas de formaldehído exógeno (5 mM y 2 mM), mostró un incremento del 20% y 30 % con respecto a plantas de Arabidopsis sin transformar. Aunque la sobreexpresión del gen, sin embargo, no impide que la exposición al formaldehído (5 mM) durante 24 H tendrá un efecto citotóxico, que produce la destrucción de los cloroplastos. De otra parte, la inmunolocalización de la FALDH en células de tabaco BY2, mostró que se encuentra en el citoplasma como en el núcleo. Su distribución es similar a la de la _-tubulina en las diferentes etapas del ciclo celular.Formaldehyde is cytotoxic agent that provokes mutations in DNA, and has been categorised as a carcinogen. It is produced during normal cellular metabolism, but its concentration increases by oxidative stress. Formaldehyde is also an important atmospheric pollant and a contaminant of risidual industrial waters. The main enzymatic system metabolising formaldehyde in eukaryotes is the gluthione-dependent formaldehyde dehydrogenase. The glutathione-dependent formaldehyde dehyrogenase (FALDH), also known as class III ADH, catalyzes the NAD+-dependent oxidation of formaldehyde. The true substrate of enzyme in this reaction is the S-hydroxymethylglutathione that forms spontaneously between formaldehyde and glutathione .The enzyme has a ubiquitous distribution in animals and plants, with constitutive levels of expresión, It has long been proposed that the primary function of this enzyme is to detoxify endogenous and exogenous formaldehyde. We have investigated the effects of overexpressing the FALDH gene in two living organisms, Saccharomyces cerevisiae and Arabidopsis thaliana. Overexpressing in yeatst confers high resístanse to formadehyde since the transformed strain is able to grow much better than the wild type strain at moderately hig concentration of this compound (2mM). Overexpressing in plants significantly increases their ability to eliminate high concentration of formaldeyde added exogeneeously to 20% and 30% en comparation with wild type. The rate of formaldehyde elimination is directly related to the amount of FALDH activity expressed by the plant. Tus confirming the essential role of this enzyme in the metabolism of formaldehyde. However, exposure to very high concentrations of formaldehyde (5mM) produces important phytotoxic effects both in the wild type and in transgenic arabidopsis plants,wich start to disply chlorotic lesions in the leaves after 24h of exposure. Examination by electronic microscopy revealed that those lesions were due to massive destruction of chloroplasts. These results open important issues for biotechnological aplications in the bioremediation of environmental formaldehyde. Also, we studied the subcellular localization of the enzyme in de tabacco BY2 cell line, and we demostrated the localization of FALDH in the cytoplasme and in the nucleus. The distribution of FALDH is identic to _-tubuline in diferentes phases of the cell cycle

Estudio funcional de la formoldehido deshidrogenasa dependiente de glutation de arobidopsis thaliana. Aplicaciones en fitoremediación

El formaldehído es un agente citotóxico que provoca mutaciones in el DNA, ha sido clasificado como un carcinógeno. Se produce durante el metabolismo celular normal, pero su concentración aumenta con el estrés oxidativo. El formaldehído es un polucionante atmosférico (especialmente de interiores) y de aguas residuales. El principal sistema enzimático que metaboliza el formaldehído en los organismos eucariotas es mediante la formaldehído deshidrogenasa dependiente de glutatión. La formaldehído dehidrogenasa dependiente de glutatión, también, llamada ADH clase III, en presencia del NAD+ cataliza la oxidación de formaldehído. El sustrato real del enzima es el S-hidroximetilglutatión que se forma de la reacción espontánea entre el formaldehído y el glutatión. La FALDH se encuentra universalmente distribuida en todos los organismos, tanto en el reino vegetal como animal. Ambas propiedades del enzima, distribución ubicua y alta conservación de secuencia, sugieren que la función principal de este enzima es metabolismo del formaldehído tanto endógeno como exógeno.Se ha estudiado el efecto de sobreexpresión del gen de la FALDH en dos organismos Sacaromyces cerevisiae y Arabidopsis thaliana. La sobre expresión en un vector multicopias del gen FALDH en levadura confiere a ésta la capacidad de resistir a altas concentraciones de formaldehído (2 mM). También, se han obtenido varias plantas transgénicas que sobreexpresan la FALDH de Arabidopsis. El estudio de la capacidad de las diferentes líneas transgénicas de metabolizar concentraciones altas de formaldehído exógeno (5 mM y 2 mM), mostró un incremento del 20% y 30 % con respecto a plantas de Arabidopsis sin transformar. Aunque la sobreexpresión del gen, sin embargo, no impide que la exposición al formaldehído (5 mM) durante 24 H tendrá un efecto citotóxico, que produce la destrucción de los cloroplastos.De otra parte, la inmunolocalización de la FALDH en células de tabaco BY2, mostró que se encuentra en el citoplasma como en el núcleo. Su distribución es similar a la de la _-tubulina en las diferentes etapas del ciclo celular.Formaldehyde is cytotoxic agent that provokes mutations in DNA, and has been categorised as a carcinogen. It is produced during normal cellular metabolism, but its concentration increases by oxidative stress. Formaldehyde is also an important atmospheric pollant and a contaminant of risidual industrial waters. The main enzymatic system metabolising formaldehyde in eukaryotes is the gluthione-dependent formaldehyde dehydrogenase. The glutathione-dependent formaldehyde dehyrogenase (FALDH), also known as class III ADH, catalyzes the NAD+-dependent oxidation of formaldehyde. The true substrate of enzyme in this reaction is the S-hydroxymethylglutathione that forms spontaneously between formaldehyde and glutathione .The enzyme has a ubiquitous distribution in animals and plants, with constitutive levels of expresión, It has long been proposed that the primary function of this enzyme is to detoxify endogenous and exogenous formaldehyde.We have investigated the effects of overexpressing the FALDH gene in two living organisms, Saccharomyces cerevisiae and Arabidopsis thaliana. Overexpressing in yeatst confers high resístanse to formadehyde since the transformed strain is able to grow much better than the wild type strain at moderately hig concentration of this compound (2mM). Overexpressing in plants significantly increases their ability to eliminate high concentration of formaldeyde added exogeneeously to 20% and 30% en comparation with wild type. The rate of formaldehyde elimination is directly related to the amount of FALDH activity expressed by the plant. Tus confirming the essential role of this enzyme in the metabolism of formaldehyde. However, exposure to very high concentrations of formaldehyde (5mM) produces important phytotoxic effects both in the wild type and in transgenic arabidopsis plants,wich start to disply chlorotic lesions in the leaves after 24h of exposure. Examination by electronic microscopy revealed that those lesions were due to massive destruction of chloroplasts. These results open important issues for biotechnological aplications in the bioremediation of environmental formaldehyde.Also, we studied the subcellular localization of the enzyme in de tabacco BY2 cell line, and we demostrated the localization of FALDH in the cytoplasme and in the nucleus. The distribution of FALDH is identic to _-tubuline in diferentes phases of the cell cycle

Enhanced Formaldehyde Detoxification by Overexpression of Glutathione-Dependent Formaldehyde Dehydrogenase from Arabidopsis

The ADH2 gene codes for the Arabidopsis glutathione-dependent formaldehyde dehydrogenase (FALDH), an enzyme involved in formaldehyde metabolism in eukaryotes. In the present work, we have investigated the potential role of FALDH in detoxification of exogenous formaldehyde. We have generated a yeast (Saccharomyces cerevisiae) mutant strain (sfa1Δ) by in vivo deletion of the SFA1 gene that codes for the endogenous FALDH. Overexpression of Arabidopsis FALDH in this mutant confers high resistance to formaldehyde added exogenously, which demonstrates the functional conservation of the enzyme through evolution and supports its essential role in formaldehyde metabolism. To investigate the role of the enzyme in plants, we have generated Arabidopsis transgenic lines with modified levels of FALDH. Plants overexpressing the enzyme show a 25% increase in their efficiency to take up exogenous formaldehyde, whereas plants with reduced levels of FALDH (due to either a cosuppression phenotype or to the expression of an antisense construct) show a marked slower rate and reduced ability for formaldehyde detoxification as compared with the wild-type Arabidopsis. These results show that the capacity to take up and detoxify high concentrations of formaldehyde is proportionally related to the FALDH activity in the plant, revealing the essential role of this enzyme in formaldehyde detoxification