Chloroplasts as a nitric oxide cellular source : Effect of reactive nitrogen species on chloroplastic lipids and proteins

Nitric oxide (NO) generation by soybean (Glycine max var. ADM 4800) chloroplasts was studied as an endogenous product assessed by the electron paramagnetic resonance spin-trapping technique. Nitrite and L-arginine (Arg) are substrates for enzymatic activities considered to be the possible sources of NO in plants. Soybean chloroplasts showed a NO production of 3.2 ± 0.2 nmol min-1 mg-1 protein in the presence of 1 mM NaNO 2. Inhibition of photosynthetic electron flow by 3-(3,4- dichlorophenyl)-1,1-dimethyl urea resulted in a lower rate (1.21 ± 0.04 nmol min-1 mg-1 protein) of NO generation. Chloroplasts incubated with 1 mM Arg showed NO production of 0.76 ± 0.04 nmol min -1 mg-1 protein that was not affected either by omission of Ca2+ or by supplementation with Ca2+ and calmodulin to the incubation medium. This production was inhibited when chloroplasts were incubated in the presence of NO synthase inhibitors Nω,-nitro- L-Arg methyl ester hydrochloride and Nω-nitro-L-Arg. In vitro exposure of chloroplasts to an NO donor (250 μM S-nitrosoglutathione) decreased lipid radical content in membranes by 29%; however, incubation in the presence of 25 μM peroxynitrite (ONOO-) led to an increase in lipid-derived radicals (34%). The effect of ONOO- on protein oxidation was determined by western blotting, showing an increase in carbonyl content either in stroma or thylakoid proteins as compared to controls. Moreover, ONOO- treatment significantly affected both O2 evolution and chlorophyll fluorescence in thylakoids. Data reported here suggest that NO is an endogenous metabolite in soybean chloroplasts and that reactive nitrogen species could exert either antioxidant or prooxidant effects on chloroplast macromolecules.Instituto de Fisiología Vegeta

Chloroplasts as a nitric oxide cellular source : Effect of reactive nitrogen species on chloroplastic lipids and proteins

Nitric oxide (NO) generation by soybean (Glycine max var. ADM 4800) chloroplasts was studied as an endogenous product assessed by the electron paramagnetic resonance spin-trapping technique. Nitrite and L-arginine (Arg) are substrates for enzymatic activities considered to be the possible sources of NO in plants. Soybean chloroplasts showed a NO production of 3.2 ± 0.2 nmol min-1 mg-1 protein in the presence of 1 mM NaNO 2. Inhibition of photosynthetic electron flow by 3-(3,4- dichlorophenyl)-1,1-dimethyl urea resulted in a lower rate (1.21 ± 0.04 nmol min-1 mg-1 protein) of NO generation. Chloroplasts incubated with 1 mM Arg showed NO production of 0.76 ± 0.04 nmol min -1 mg-1 protein that was not affected either by omission of Ca2+ or by supplementation with Ca2+ and calmodulin to the incubation medium. This production was inhibited when chloroplasts were incubated in the presence of NO synthase inhibitors Nω,-nitro- L-Arg methyl ester hydrochloride and Nω-nitro-L-Arg. In vitro exposure of chloroplasts to an NO donor (250 μM S-nitrosoglutathione) decreased lipid radical content in membranes by 29%; however, incubation in the presence of 25 μM peroxynitrite (ONOO-) led to an increase in lipid-derived radicals (34%). The effect of ONOO- on protein oxidation was determined by western blotting, showing an increase in carbonyl content either in stroma or thylakoid proteins as compared to controls. Moreover, ONOO- treatment significantly affected both O2 evolution and chlorophyll fluorescence in thylakoids. Data reported here suggest that NO is an endogenous metabolite in soybean chloroplasts and that reactive nitrogen species could exert either antioxidant or prooxidant effects on chloroplast macromolecules.Instituto de Fisiología Vegeta

Exposure to nitric oxide protects against oxidative damage but increases the labile iron pool in sorghum embryonic axes

Sodium nitroprusside (SNP) and diethylenetriamine NONOate (DETA NONOate), were used as the source of exogenous NO to study the effect of NO upon germination of sorghum (Sorghum bicolor (L.) Moench) seeds through its possible interaction with iron. Modulation of cellular Fe status could be an important factor for the establishment of oxidative stress and the regulation of plant physiology. Fresh and dry weights of the embryonic axes were significantly increased in the presence of 0.1 mM SNP, as compared to control. Spin trapping EPR was used to assess the NO content in axes from control seeds after 24 h of imbibition (2.4±0.2 nmol NO g−1 FW) and seeds exposed to 0.01, 0.1, and 1 mM SNP (3.1±0.3, 4.6±0.2, and 6.0±0.9 nmol NO g−1 FW, respectively) and 1 mM DETA NONOate (6.2±0.6 nmol NO g−1 FW). Incubation of seeds with 1 mM SNP protected against oxidative damage to lipids and maintained membrane integrity. The content of the deferoxamine–Fe (III) complex significantly increased in homogenates of axes excised from seeds incubated in the presence of 1 mM SNP or 1 mM DETA NONOate as compared to the control (19±2 nmol Fe g−1 FW, 15.2±0.5 nmol Fe g−1 FW, and 8±1 nmol Fe g−1 FW, respectively), whereas total Fe content in the axes was not affected by the NO donor exposure. Data presented here provide experimental evidence to support the hypothesis that increased availability of NO drives not only protective effects to biomacromolecules, but to increasing the Fe availability for promoting cellular development as well

Chloroplasts as a Nitric Oxide Cellular Source. Effect of Reactive Nitrogen Species on Chloroplastic Lipids and Proteins

Nitric oxide (NO) generation by soybean (Glycine max var. ADM 4800) chloroplasts was studied as an endogenous product assessed by the electron paramagnetic resonance spin-trapping technique. Nitrite and l-arginine (Arg) are substrates for enzymatic activities considered to be the possible sources of NO in plants. Soybean chloroplasts showed a NO production of 3.2 ± 0.2 nmol min(−1) mg(−1) protein in the presence of 1 mm NaNO(2). Inhibition of photosynthetic electron flow by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea resulted in a lower rate (1.21 ± 0.04 nmol min(−1) mg(−1) protein) of NO generation. Chloroplasts incubated with 1 mm Arg showed NO production of 0.76 ± 0.04 nmol min(−1) mg(−1) protein that was not affected either by omission of Ca(2+) or by supplementation with Ca(2+) and calmodulin to the incubation medium. This production was inhibited when chloroplasts were incubated in the presence of NO synthase inhibitors N(ω)-nitro-l-Arg methyl ester hydrochloride and N(ω)-nitro-l-Arg. In vitro exposure of chloroplasts to an NO donor (250 μm S-nitrosoglutathione) decreased lipid radical content in membranes by 29%; however, incubation in the presence of 25 μm peroxynitrite (ONOO(−)) led to an increase in lipid-derived radicals (34%). The effect of ONOO(−) on protein oxidation was determined by western blotting, showing an increase in carbonyl content either in stroma or thylakoid proteins as compared to controls. Moreover, ONOO(−) treatment significantly affected both O(2) evolution and chlorophyll fluorescence in thylakoids. Data reported here suggest that NO is an endogenous metabolite in soybean chloroplasts and that reactive nitrogen species could exert either antioxidant or prooxidant effects on chloroplast macromolecules