Cell Localization of DPI-Dependent Production of Superoxide in Reproductive Tissues of the Olive Tree (Olea europaea L.)

Abstract

Reactive Oxygen Species (ROS) are compounds derived from oxygen with important implications in biological processes in plants, some of them related to reproduction. Among ROS, superoxide is the primary oxidant, since an array of other ROS are eventually derived from this anion. Therefore, analysis of the molecular systems able to generate this molecule and the cellular compartmentalization of these events is of paramount importance. We have used the fluorochrome DCFH2-DA and the chromogenic substrate NBT in association with DPI (a specific inhibitor of Rboh enzymes generating superoxide in plants) in combination with confocal microscopy and stereomicroscopy, respectively to identify cell localization of ROS in general, and superoxide accumulation in olive reproductive tissues. A significant production of both ROS and superoxide has been described, showing a fairly precise spatial and temporal location throughout olive flower development. The reduction of the NBT signal after the addition of DPI suggests that the generation of superoxide is largely due to Rboh or other flavin oxidase activity. At the subcellular level, accumulation of O2●− has been located in the plasma membrane of mature pollen and germinated pollen, as well as in the rough endoplasmic reticulum and in mitochondria.