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

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Abstract
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 NaNO2. 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 N ω-nitro-l-Arg methyl ester hydrochloride and N ω-nitro-l-Arg. In vitro exposure of chloroplasts to an NO donor (250 μ m S-nitrosoglutathione) decreased lipid radical content in membranes by 29%; however, incubation in the presence of 25 μ m 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.