Ogawa K, Kanematsu S, Asada K
Department of Botany, Faculty of Science, Kyoto University, Japan.
Plant Cell Physiol. 1997 Oct;38(10):1118-26. doi: 10.1093/oxfordjournals.pcp.a029096.
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.
通过分别用氮蓝四唑（NBT）和淀粉 - 碘化物染色，确定了菠菜幼苗下胚轴横切面中超氧阴离子和过氧化氢的产生部位。超氧阴离子将NBT还原生成的甲臜，仅在存在铜锌超氧化物歧化酶（CuZn - SOD）抑制剂的情况下主要在维管组织中观察到，并且在厌氧条件下其形成受到抑制。因此，NBT被维管组织中产生的超氧阴离子特异性还原为甲臜。NBT的还原受到NAD(P)H氧化酶抑制剂的抑制，但不受氰化物或叠氮化物的抑制，这表明NAD(P)H氧化酶参与了维管组织中超氧阴离子的产生。淀粉 - I₂ 复合物也在维管组织中形成，但在存在CuZn - SOD抑制剂或NAD(P)H氧化酶抑制剂的情况下则不会形成，这表明过氧化氢是通过NAD(P)H氧化酶产生的超氧阴离子与CuZn - SOD的催化歧化作用产生的。维管组织中超氧阴离子和过氧化氢的产生在木质部中尤为明显，并且与通过免疫组织化学方法确定的CuZn - SOD的分布部位相关，也与通过间苯三酚 - 盐酸反应确定的木质素的位置相关。
