Generation of superoxide anion and localization of CuZn-superoxide dismutase in the vascular tissue of spinach hypocotyls: their association with lignification.

The sites of generations of superoxide anions and hydrogen peroxide in cross sections of hypocotyls from spinach seedlings were located by staining with nitroblue tetrazolium (NBT) and with starch-iodide, respectively. Formazan, produced upon the reduction of NBT by superoxide, was observed mainly in the vascular tissue only in the presence of inhibitors of CuZn-superoxide dismutase (CuZn-SOD), and its formation was suppressed under anaerobic conditions. Thus, NBT was reduced to formazan specifically by the superoxide anions generated in vascular tissue. The reduction of NBT was suppressed by inhibitors of NAD(P)H oxidase, but neither by cyanide nor azide, indicating the involvement of NAD(P)H oxidase in the generation of superoxide anions in the vascular tissue. Starch-I2 complex also was formed in the vascular tissue, but not in the presence of either the CuZn-SOD inhibitor or the NAD(P)H oxidase inhibitor, indicating that the hydrogen peroxide is produced via the catalytic disproportionation with CuZn-SOD of the superoxide generated by NAD(P)H oxidase. Generations of superoxide anions and hydrogen peroxide in the vascular tissue were particularly apparent in the xylem and associated with the sites of distribution of CuZn-SOD as determined by an immunohistochemical method, and also with the location of lignin as determined by the phloroglucin-HCl reaction.