Preparation of mitochondria to measure superoxide flashes in angiosperm flowers

Abstract

Background. Mitochondria are the center of energy metabolism and the production of reactive oxygen species (ROS). ROS production results in a burst of superoxide flashes", which is always accompanied by depolarization of mitochondrial membrane potential. Superoxide flashes have only been studied in the model plant Arabidopsis thaliana using a complex method to isolate mitochondria. In this study, we present an efficient, easier method to isolate functional mitochondria from floral tissues to measure superoxide flashes. Method. We used 0.5 g samples to isolate mitochondria within <1.5 h from flowers of two non-transgenic plants (Magnolia denudata and Nelumbo nucifera) to measure superoxide flashes. Superoxide flashes were visualized by the pH-insensitive indicator MitoSOX Red, while the mitochondrial membrane potential (19 m) was labelled with TMRM. Results. Mitochondria isolated using our method showed a high respiration ratio. Our results indicate that the location of ROS and mitochondria was in a good coincidence. Increased ROS together with a higher frequency of superoxide flashes was found in mitochondria isolated from the flower pistil. Furthermore, a higher rate of depolarization of the 19 m was observed in the pistil. Taken together, these results demonstrate that the frequency of superoxide flashes is closely related to depolarization of the 19 m in petals and pistils of flowers.