GENERATION OF SUPEROXIDE ANION AND LOCALIZATION OF CUZN-SUPEROXIDE DISMUTASE IN THE VASCULAR TISSUE OF SPINACH HYPOCOTYLS - THEIR ASSOCIATION WITH LIGNIFICATION

The sides of generations of superoxide anions and hydrogen peroxide in cross sections of hypocotyls from spinach seedlings were located by s taining with nitroblue tetrazolium (NBT) and with starch-iodide, respe ctively, Formazan, produced upon the reduction of NBT by superoxide, w as observed mainly in the vascular tissue only ire the presence of inh ibitors of CuZn-superoxide dismutase (CuZn-SOD), and its formation was suppressed under anaerobic conditions. Thus, NBT was reduced to forma zan specifically by the superoxide anions generated in vascular tissue . The reduction of NBT was suppressed by inhibitors of NAD(P)H oxidase , hut neither by cyanide nor azide, indicating the involvement of NAD( P)H oxidase in the generation of superoxide anions in the vascular tis sue, Starch-I-2 complex also was formed in the vascular tissue, but no t in the presence of either the CuZn-SOD inhibitor or the NAD(P)PI oxi dase inhibitor, indicating that the hydrogen peroxide is produced via the catalytic disproportionation with CuZn-SOD of the superoxide gener ated by NAD(P)H oxidase. Generations of superoxide anions and hydrogen peroxide in the vascular tissue were particularly apparent in the xyl em and associated with the sites of distribution of CuZn-SOD as determ ined by an immunohistochemical method, and also with the location of l ignin as determined bg the phloroglucin-HCl reaction.