NADH-stimulated, cyanide-resistant superoxide production in maize coleoptiles analyzed with a tetrazolium-based assay
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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 (O2·−) production in vivo, we have shown that maize (Zea mays L.) coleoptiles possess the capacity of generating O2·− 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 O2·−-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 O2·−-producing activity of coleoptiles. The maize NADH-dependent O2·−-synthase activity could clearly be differentiated from peroxidase-mediated O2·−-synthesizing activity by its insensitivity to cyanide and azide, as well as by its much higher affinity to O2. Formation of O2·−, and concomitantly appearing H2O2, was preferentially localized in the outer epidermis of the coleoptile. The physiological significance of O2·− and H2O2 production in relation to the growth-controlling function of the epidermal cell wall is discussed.
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