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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Chloroplasts as a Nitric Oxide Cellular Source. Effect of Reactive Nitrogen Species on Chloroplastic Lipids and Proteins¹

Physical Chemistry-PRALIB, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina (S.J., M.S., S.P.); and Instituto de Fisiología Vegetal, Facultad de Ciencias Naturales, Universidad Nacional de La Plata, La Plata, Argentina (C.G.B.)

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-nitro-L-Arg methyl ester hydrochloride and N-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.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Susana Puntarulo (susanap{at}ffyb.uba.ar).

www.plantphysiol.org/cgi/doi/10.1104/pp.106.086918

^* Corresponding author; e-mail susanap{at}ffyb.uba.ar; fax 54–11–4508–3646.

Received July 18, 2006; accepted September 11, 2006; published September 15, 2006.

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