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Besides providing energy to sustain life, mitochondria also play crucial roles in stress response and programmed cell death. The mitochondrial hallmark lipid, cardiolipin (CL), is essential to the maintenance of mitochondrial structure and function. However, how mitochondria and CL are involved in stress response is not as well defined in plants as in animal and yeast cells. We previously revealed a role for CL in mitochondrial fission and in heat stress response in Arabidopsis. To further determine the involvement of mitochondria and CL in plant heat response, here we treated Arabidopsis seedlings with varied lengths of acute heat stress. These treatments resulted in decreases in mitochondrial membrane potential, disruption of mitochondrial ultrastructure, accumulation of mitochondrial reactive-oxygen species (ROS), and redistribution of CL to the outer mitochondrial membrane and to a novel type of vesicle. The …