Reactive Nitrogen Species-Dependent Effects on Soybean Chloroplasts
2007
Puntarulo, Susana | Jasid, Sebastián | Simontacchi, Marcela
Nitric oxide (NO) generation by soybean (Glycine max, var ADM 4800) chloroplasts was studied by electron paramagnetic resonance (EPR) spin-trapping technique1. Both nitrite and L-arginine (arg) are the required substrates for enzymatic activities considered as 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. Chloroplasts incubated with 1 mM arg showed a NO production of 0.76 ± 0.04 nmol min-1 mg-1 protein. This production was inhibited when chloroplasts were incubated in presence of NOS-inhibitors L-NAME and L-NNA. In vitro exposure of chloroplasts to a NO-donor (GSNO) decreased both ascorbyl radical content and the activity of ascorbate peroxidase, without modification of the total ascorbate content. Exposure of the isolated chloroplasts to a NO-donor decreased lipid radical content in membranes, 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 control. Taken as a whole, NO seems to be an endogenous metabolite in soybean chloroplasts and reactive nitrogen species could exert either antioxidant or prooxidant effects on chloroplasts, since both a decreased lipid radical content in membranes and a decrease in the activity of ascorbate peroxidase were observed after exposure to a NO donor.
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