NADH-stimulated, cyanide-resistant superoxide production in maize coleoptiles analyzed with a tetrazolium-based assay

Using the tetrazolium salt XTT (Na,3'-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate) as a sensitive and physiologically compatible probe for the determination of superoxide (O-2(.-)) production in vivo, we have shown that maize (Zen mays L.) coleoptiles possess the capacity of generating O-2(.-) in the apoplastic space. Our results are in agreement with the notion that this activity is localized at the plasma membrane and can be attributed to an O-2(.-)-synthesizing enzyme with catalytic and kinetic properties similar to that of the NADPH oxidase of mammalian phagocytes, with the important exception that it utilizes NADH instead of NADPH as electron donor. When applied to the apoplastic space, NADH strongly increased the O-2(.-)-producing activity of coleoptiles. The maize NADH-dependent O-2(.-)-synthase activity could clearly be differentiated from peroxidase-mediated O-2(.-)-synthesizing activity by its insensitivity to cyanide and azide, as well as by its much higher affinity to O-2 Formation of O-2(.-), and concomitantly appearing H2O2, was preferentially localized in the outer epidermis of the coleoptile. The physiological significance of O-2(.-) and H2O2 production in relation to the growth-controlling function of the epidermal cell wall is discussed.