Reactive Nitrogen Species Generation and Effects on Leaves Chloroplasts.

MARCELA SIMONTACCHI; SEBASTIÁN JASID; SUSANA PUNTARULO

Rosario

Congreso; XXXV Reunión Anual de la Sociedad Argentina de Biofísica; 2006

Sociedad Argentina de Biofísica

Nitric oxide (NO) is both a gaseous free radical and a bioactive molecule that plays important roles in diverse processes in plants [1,2]. Since NO was identified as an endogenous metabolite, it has been proposed to have either antioxidant or prooxidant effects [3,4]. It is believed that while NO by itself is involved in protective functions [5], the deleterious effects could be due to peroxynitrite anion (ONOO-), a species with a potent oxidizing power [6]. In this work, soybean (Glycine max, var ADM 4800) chloroplasts were incubated in vitro with an NO-donor (250 mM GSNO). This treatment produced a decrease in lipid radical content in chloroplast membranes by 29%, however, incubation in the presence of 25 mM 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. NO production by plant tissues has been known for some time. It has been established that plants can generate NO by enzymatic [7, 8] and non-enzymatic ways [9]. Within the enzymatic pathways nitrite or L-arginine (Arg) are the substrates for these activities considered to be the possible sources of NO in plants. Nitric oxide generation by chloroplasts was assessed by electron paramagnetic resonance (EPR) spin-trapping technique. 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. Thylakoid membranes showed a lower rate of NO generation as compared to intact chloroplasts, whereas isolated stroma showed a negligible rate of NO synthesis. 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. 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 in a way strongly dependent on the oxidative environment.   [1] Neill SJ, Desikan R, Clarke A, Hurst RD and Hancock JT, J Exp Bot, 2002, 53, 1237-1247. [2] Lamattina L, Garcia-Matta C, Graziano M and Pagnussat G, Annu Rev Plant Biol, 2003, 54, 109-136. [3] Caro A and Puntarulo S, Physiol Plant, 1998, 104, 357-364. [4] Beligni MV and Lamattina L, Trends Plant Sci, 1999, 4, 299-300. [5] Wink DA, Cook JA, Pacelli R, Liebmann J, Krishne MC and Mitchell JB, Toxicol Lett, 1995, 82/83, 221-226. [6] Blough NV and Zafiriou DC, Inorg Chem, 1985, 24, 3504-3505. [7] del Río LA, Corpas FJ, Barroso JB, Phytochemistry, 2004, 65, 783-792. [8] Simontacchi M, Jasid S and Puntarulo S, Plant Sci, 2004, 167, 839-847. [9] Bethke PC, Badger MR and Jones RL, Plant Cell, 2004, 16, 332-341.   Supported by grants from CONICET, UBA and ANPCyT