Decrease in mitochondrial DNA and concurrent increase in plastid DNA in generative cells of Pharbitis nil during pollen development

Abstract

The amount of organellar DNA in a generative cell of Pharbitis nil was observed when squashed pollen grains collected on the day of flowering were stained with the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole (DAPI). Using both DAPI-fluorescence microscopy and electron microscopy, observation of the same thin section of Technovit 7100 resin-embedded material revealed that all of the organellar DNA in mature generative cells is plastid DNA, and there is no mitochondrial DNA. During pollen development, we observed organellar DNA in fluorescence microscopic images using double-staining with DAPI and 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and quantified the DNA using a video-intensified microscope photon counting system (VIMPCS). In the vegetative cells, the amounts of both mitochondrial and plastid DNA progressively decreased and had disappeared by 2 days before flowering. In the generative cells, mitochondrial DNA disappeared sooner than in the vegetative cells, indicating a more active mechanism for the decrease in mitochondrial DNA in the generative cells. In contrast, plastid DNA in the generative cells increased markedly. The DNA content per plastid was at a minimum value (corresponding to one copy of the plastid genome) 7 days before flowering, but it increased to a maximum value (corresponding to over 10 copies of the plastid genome) 2 days before flowering. Similar results were also obtained with immunogold electron microscopy using an anti-DNA antibody. These results suggest that the DNA content of mitochondria and plastids in P. nil is controlled independently during pollen development.