Zhang Chulan, Sun Fengshuo, Xiong Biao, Zhang Zhixiang
College of Nature Conservation, Beijing Forestry University, Beijing, China.
College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing, China.
PeerJ. 2019 Apr 11;7:e6708. doi: 10.7717/peerj.6708. eCollection 2019.
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 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.
We used 0.5 g samples to isolate mitochondria within <1.5 h from flowers of two non-transgenic plants ( and ) 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.
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.
线粒体是能量代谢和活性氧（ROS）产生的中心。ROS的产生会导致一阵“超氧阴离子闪光”，这总是伴随着线粒体膜电位的去极化。超氧阴离子闪光仅在模式植物中使用复杂的线粒体分离方法进行过研究。在本研究中，我们提出了一种从花组织中分离功能性线粒体以测量超氧阴离子闪光的高效、简便方法。
我们使用0.5克样本，在不到1.5小时的时间内从两种非转基因植物的花朵中分离线粒体以测量超氧阴离子闪光。超氧阴离子闪光通过对pH不敏感的指示剂MitoSOX Red可视化，而线粒体膜电位（ΔΨm）用TMRM标记。
使用我们的方法分离的线粒体显示出高呼吸率。我们的结果表明ROS和线粒体的位置吻合良好。在从花雌蕊分离的线粒体中发现ROS增加以及超氧阴离子闪光频率更高。此外，在雌蕊中观察到ΔΨm的去极化速率更高。综上所述，这些结果表明超氧阴离子闪光频率与花朵花瓣和雌蕊中ΔΨm的去极化密切相关。
