The DNA in isolated chloroplasts was visualized by the fluorescent probe 4'6-diamidino-2-phenylindole (DAPI). When excited with light of 360 nm, the DNA-DAPI complex fluoresces brilliantly at 450 rim. Nuclei also fluoresce but their nucleoli do not. RNase and Pronase treatment of chloroplasts did not affect the fluorescence but both pre-and posttreatment of DAPI-stained chloroplasts with DNase specifically destroyed the fluorescence. DNA-DAPI complexes in the chloroplasts show up as bright dots. These are distributed uniformly within the chloroplast except for the outer margins. The fluorescent dots can be seen at different focal levels. The number of DNA dots is roughly proportional to chloroplast area which, in turn, is a function of leaf size. The number of fluorescent dots also gave the impression that large leaves with large chloroplasts contain more chloroplast DNA than nuclear DNA.KEY WORDS higher plant chloroplast DNA DAPI-DNA complex fluorescence microscopy granaThe existence of DNA in cell organelles other than nuclei evokes questions such as: how much extranuclear DNA is present per cell; how much is present per mitocbondrion or chloroplast; how is it duplicated; and how is it arranged relative to the molecular architecture of the organelle? For chloroplasts, an answer to the last question would be greatly facilitated by a methodology whereby DNA could be traced from the level of light microscopy to the details of construction which have been sought by electron microscopy (11,13,20,21). A discovery by Williamson and Fennell (19) has made possible the optical localization of organelle DNA. They showed that a trypanocide, 4'6-diamidino-2-phenylindole (DAPI), synthesized by Dann et al. (2), is a probe capable of revealing mitochondrial DNA in yeast by fluorescence microscopy. Subsequent studies have employed DAPI as a probe to visualize mycoplasmas in tissue culture cells (14) and kinetoplast DNA in trypanosomes (3).Previous studies employed acridine orange as a fluorescence probe for DNA in plastids (13) and chloroplasts (20) of algal forms. In this report, we show that DAPI is both more intense and more specific as a fluorescence probe for DNA in nuclei and chloroplasts isolated from leaves of higher plants than acridine orange. The specificity of DAPI binding to DNA has provided a means to see hitherto unknown details of the organization, character, and relative amounts of DNA per chloroplast. MATERIALS AND METHODS Plant MaterialTobacco ( Nicotiana tabacum ), spinach (Spinacea oleracea), and sugar beet (Beta vulgaris alba) were grown J. CELL BIOLOGY 9 The Rockefeller University Press

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Cell-free homogenates containing intact chloroplasts and nuclei were allowed to settle for up to 1 h before the top 2 ml of the 5-ml homogenate was withdrawn. Whereas less than 18% of the chloroplasts moved from the top to the bottom portions, the ratio of nuclei to chloroplasts in the top portion changed from approximately 1/200 to 1/900. The total numbers of chloroplasts and nuclei were counted in the homogenate before settling and in the top 2 ml and bottom 3 m1 after settling. The total DNA content of the homogenate and the top and bottom portions after settling was determined by the diphenylamine colorimetric assay. By simultaneous equations, the absolute amount of DNA in chloroplasts and nuclei was determined. The results are consistent with previous observations of chloroplast DNA by fluorescence microscopy which indicated that the amount of chloroplast DNA per chloroplast is a function of chloroplast size. In addition, the results show that the amount of chloroplast DNA per average chloroplast in large leaves is 0.14 times 10(-12) g, a magnitude higher than previous reports in the literature, and that large leaves contain about twice as much chloroplast DNA as nuclear DNA.

By phase microscopic observation of living palisade parenchyma cells in sections of Nicotiana excelsior leaves from plants previously placed in the dark for 72 hours, 30 to 45 minutes of light is found to induce mitochondria to remain stationary within the concavity of the chloroplasts and become round. Extending the illumination period to 60 to 90 minutes causes the stationary mitochondria in the concavity to change from a translucent to an opaque appearance, the change coinciding with the first appearance of starch as detected by blue staining of the grains with I2-KI. It is speculated that an interaction bearing some resemblance to the previously described interaction between mitochondria and the mobile phase of the chloroplasts may also operate in the starch grain phenomenon.In previous reports, an interaction between mitochondria and the mobile phase of chloroplasts has been described (1,3,4) and recorded by cinemicrophotography.' This interaction consists of mobile phase protuberances segmenting into free streaming organelles which cannot be distinquished from other free streaming mitochondria in the living cell. Conversely, the interaction involves a free streaming mitochondrion coalescing with the mobile phase followed by complete loss of identity of the mitochondrion after the act of fusion. We have now observed another kind of chloroplast-mitochondrion interaction which concerns the origin of chloroplast starch grains which is the subject of this paper.MATERIALS AND METHODS Plant Material. Nicotiana excelsior was grown in a greenhouse. Leaves in positions 5, 6, and 7 above the cotyledons, ranging in length from 10 to 12 cm were used for experiments.

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