Nadph oxidase involvement in cd-induced oxidative stress in sunflower leaf discs

M.P. IANUZZO; M.D. GROPPA; M.P. BENAVIDES

Pinamar, Buenos Aires Argentina

Congreso; -X Congress of the Panamerican Association for Biochemistry and Molecular Biology (PABMB). XLI Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology (SAIB).XX Annual Meeting of the Argentine Society for Neurochemistry (SAN).; 2005

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

A rapid generation of reactive oxygen species (ROS), mainly superoxide anion (O2.-) and H2O2 are considered to be a component of the intracellular response to biotic and abiotic stress. The likely source of ROS is a NADPH oxidase originally postulated as a membrane-bound enzyme. The objective of the present work was to elucidate which type of ROS mediates the signaling pathways involved in cadmium toxicity in sunflower leaf discs.treated with 0.5 mM CdCl2 as well as the role of a plant NADPH oxidase in ROS formation. Superoxide anion formation was analyzed using NBT while H2O2 evolution was studied with DAB and xylenol orange. NADPH-dependent oxidase activity was measured indirectly using the SOD-inhibited reduction of NBT, after incubation of sunflower leaf discs with several inhibitors or  inducers (100 mM DPI, 1 mM LaCl3, 10mM Cl2Ca,  1 mM EGTA). NADPH-dependent oxidase activity was inhibited by 0.5 mM Cd in the cytosolic fraction (to 27% of the C). This decay was neither reversed by LaCl3 nor by CaCl2, while EGTA showed a variable behaviour. The addition of DPI, a NADPH dependent oxidase inhibitor, to the reaction medium decreased oxidase activity by over 60% whereas NaN3 (a haem protein inhibitor) had no significant effect on superoxide generation. Hydrogen peroxide formation was inhibited by Cd 0.5 mM by 40% respect to the controls and this this inhibition was reverted by both LaCl3 and CaCl2 almost to de C values. EGTA did not avoid Cd-induced enzyme inhibition Our result suggest that superoxide and H2O2 generation in sunflower leaf discs is regulated in Cd-treated sunflower leaf discs by the inhibition of NADPH-dependent oxidase activity and the role of calcium in ROS formation seemed to be more related to H2O2 evolution than to superoxide formation