The localization of NADPH oxidase and reactive oxygen species in in vitro-cultured Mesembryanthemum crystallinum L. hypocotyls discloses their differing roles in rhizogenesis

This work demonstrated how reactive oxygen species (ROS) are involved in the regulation of rhizogenesis from hypocotyls of Mesembryanthemum crystallinum L. cultured on a medium containing 1-naphthaleneacetic acid (NAA). The increase of NADPH oxidase activity was correlated with an increase of hydrogen peroxide (H(2)O(2)) content and induction of mitotic activity in vascular cylinder cells, leading to root formation from cultured hypocotyls. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, inhibited H(2)O(2) production and blocked rhizogenesis. Ultrastructural studies revealed differences in H(2)O(2) localization between the vascular cylinder cells and cortex parenchyma cells of cultured explants. We suggest that NADPH oxidase is responsible for H(2)O(2) level regulation in vascular cylinder cells, while peroxidase (POD) participates in H(2)O(2) level regulation in cortex cells. Blue formazan (NBT) precipitates indicating superoxide radical (O(2) (•−)) accumulation were localized within the vascular cylinder cells during the early stages of rhizogenesis and at the tip of root primordia, as well as in the distal and middle parts of newly formed organs. 3,3′-diaminobenzidine (DAB) staining of H(2)O(2) was more intense in vascular bundle cells and in cortex cells. In newly formed roots, H(2)O(2) was localized in vascular tissue. Adding DPI to the medium led to a decrease in the intensity of NBT and DAB staining in cultured explants. Accumulation of O(2) (•−) was then limited to epidermis cells, while H(2)O(2) was accumulated only in vascular tissue. These results indicate that O(2) (•−) is engaged in processes of rhizogenesis induction involving division of competent cells, while H(2)O(2) is engaged in developmental processes mainly involving cell growth.