Plant Physiol. Journal of Pharmacology and Experimental Therapeutics
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Published on September 15, 2006; 10.1104/pp.106.086918

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Received July 18, 2006
Accepted September 11, 2006

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

Sebastián Jasid , Marcela Simontacchi , Carlos G. Bartoli , and Susana Puntarulo *

Physical Chemistry-PRALIB, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina
Instituto de Fisiología Vegetal, Facultad de Ciencias Naturales, Universidad Nacional de La Plata, La Plata, Argentina

* Corresponding author; email: susanap{at}ffyb.uba.ar.

Nitric oxide (NO) generation by soybean (Glycine max, var ADM 4800) chloroplasts was studied as an endogenous product assessed by electron paramagnetic resonance (EPR) spin-trapping technique. Nitrite or L-arginine (Arg) are the 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 DCMU resulted in a lower rate (1.21 ± 0.04 nmol min-1 mg-1 prot) of NO generation. Chloroplasts incubated with 1 mM Arg showed a NO production of 0.76 ± 0.04 nmol min-1 mg-1 protein that was not affected either by the omission of Ca2+ or by the supplementation with Ca2+ and calmodulin to the incubation medium. This production was inhibited when chloroplasts were incubated in presence of NOS-inhibitors L-NAME and L-NNA. In vitro exposure of chloroplasts to an NO-donor (250 µM GSNO) 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 control. 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.




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