Studies on the Behavior of Mitochondrial DNA

Abstract

The fate of mitochondrial nuclei (known as nucleoids or mt-nuclei), which contain extremely small amounts of DNA, was followed in thin sections of the root meristem of Pelargonium zonale by embedding of samples in Technovit 7100 resin and double staining with 4′-6-diamidino-2-phenylindole (DAPI) and acridine orange, in combination with light-microscopic autoradiography and microphotometry. The synthesis of cell-nuclear DNA and cell division occurs actively in the root meristem, between 150 μm and 700 μm from the tip of the root. For simplicity, cells in S phase in the cortex were selected for main analysis as the model system for examination of cell proliferation. It is estimated, on the basis of the length of the cells in longitudinal median sections, that the cells in the cortex, which are generated in the area just above the quiescent center (QC) about 150 μm from the tip, enter the elongation zone after at least five divisions. In the entire cortex, individual cells in S phase have approximately 230 mitochondria that each contain one mt-nucleus. The observation suggests that individual mitochondria divide once per mitotic cycle in the entire region of the meristem. By contrast, on the basis of incorporation of [3H]thymidine into mt-nuclei, the synthesis of mitochondrial DNA (mtDNA) occurs independently of the mitotic cycle in a restricted region just above the QC. Fluorimetry, using a video-intensified microscope photon-counting system (VIMPICS), revealed that the mtDNA content per mt-nucleus in the cells just above QC, where the synthesis of mtDNA is active, corresponds to approximately 3000 kilobase pairs (kbp) but, in the meristematic cells just below the elongation zone of the root it falls to less than 170 kbp. These findings strongly suggest that the amount of mtDNA per mitochondrion which has been synthesized in the region just above the QC is reduced stepwise as a result of continuous divisions of mitochondria in the absence of the synthesis of mtDNA. This phenomenon would explain why differentiated cells with a large vacuole in the elongation zone have mitochondria that contain only extremely small amounts of mtDNA.