Preparation of mitochondria to measure superoxide flashes in angiosperm flowers

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 (ΔΨ 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 ΔΨ m was observed in the pistil. Taken together, these results demonstrate that the frequency of superoxide flashes is closely related to depolarization of the ΔΨ m in petals and pistils of flowers.