The localization of (3H) thymidine incorporation in the DNA of replicating spinach chloroplasts by electron-microscope autoradiography

0 CSIRO Division of Horticultural Research , G.P.O. Box 350, Adelaide, South Australia 5001 , Australia Electron-microscope autoradiography has been used to obtain information on the localization of DNA labelled with pHJthymidine in chloroplasts known to be replicating and concomitantly synthesizing and segregating DNA, in cultured leaf disks. The studies were made using both Microdol-X developer and a 'compact' developer which gave a smaller grain size. About 80 % of the grains were associated with the granal membranes and with presumptive DNA regions (3-nm fibril material in clear areas). Few grains occurred in association with the chloroplast envelope. We suggest that the DNA of chloroplasts is associated with the grana lamellae and extends into the stroma. Some light-microscope autoradiographs of whole chloroplasts show spiral or helical-like labelling patterns. We interpret these patterns as demonstration of the possibility that DNA occurs along the length of a continuous lamellar membrane system. Chloroplast fractionation experiments provided data consistent with the electron-microscope autoradiographic studies as most of the label was associated with chlorophyll-containing lamellae. - ELECTRON-MICROSCOPE AUTORADIOGRAPHY R. J. ROSE AND J. V. POSSINGHAM We consider an association of chloroplast DNA molecules along the length of a continuous lamellar system would ensure an orderly segregation of DNA to daughter chloroplasts, during the binary fission of spinach chloroplasts by constriction division. It is now well established that in the higher plant chloroplast there are many copies (20-30) of the chloroplast genome (Wells & Birnstiel, 1969; Kirk, 1972). The replicating chloroplasts of light-grown spinach leaf disks actively synthesize DNA and during the division cycle segregate it in similar amounts to the daughter chloroplasts & Rose, 1976. It is therefore possible that the chloroplast DNA (cDNA) of higher plants is located in the chloroplast in such a way as to permit such a segregation. The organization of DNA in the chloroplasts of a higher plant has been studied by of serial sections Beta vulgaris chloroplasts they found several distinctly separate DNA nucleoids, separated from each other by lamellate thylakoid stacks. As the number of nucleoids was much less than the number of chloroplast genomes, Kowallik & Herrmann (1972) R. J. Rose and J. V. Possingham concluded that each nucleoid contained 4-8 cDNA molecules. Earlier electronmicroscope studies of Swiss chard (Kislev, Swift & Bogorad, 1965) and Avena sativa (Gunning, 1965) revealed a number of DNA areas in a single chloroplast section. There appears to be no evidence to-date for a regular or definite pattern of DNA throughout higher plant chloroplasts as occurs in certain algae which have a continuous ring-shaped nucleoid lying inside the peripheral thylakoids which loop around the rim of the chloroplast (Bisalputra & Bisalputra, 1969; Gibbs, Cheng & Slankis, 1974). In these algae it appears that there are associations between the cDNA and thylakoids which facilitate this arrangement. Close associations between thylakoid membranes and DNA also occur in dinoflagellates (Kowallik & Haberkorn, 1971; Bibby & Dodge, 1974). In higher plant chloroplasts observations of cDNA membrane associations have been made in spinach (Woodcock & Fernandez-Moran, 1968) and in Pelargonium (Knoth, Herrmann, Bottger & Borner, 1974), though the regularity of this relationship and which chloroplast membranes are involved has not been clearly To obtain more information on the location of cDNA in relation to the segregation process in replicating spinach chloroplasts, we have studied the incorporation of PHJthymidine (3H-TdR) into chloroplasts using light- and electron-microscope autoradiography and chloroplast isolation and fractionation techniques. MATERIALS AND METHODS Culture of leaf disks The growth of the spinach plants (Spinacia oleracea), and subsequent leaf disk culture on sterile nutrient agar has been previously described (Possingham & Smith, 1972). Growth in the light refers to a 14 h-day, at 20000 lux, with day and night temperatures of 25 and 22 C, respectively. Growth in continuous darkness was at 25 C. The disks were incubated in 50 /iCi [63H]thymidine (27 Ci/mM, Radiochemical Centre, Amersham, U.K.), added in 1 ml under sterile conditions to the 20 ml of nutrient agar medium in 9-cm Petri dishes. The light-microscope autoradiographic techniques have been described previously, as has the specificity of incorporation of 3H-TdR into DNA (Rose et al. 1975). For electron-microscope autoradiography, tissue from the disks was fixed in 6 2 5 % glutaraldehyde, postfixed in osmium and embedded in Araldite (Cran & Possingham, 1972a). Sections were mounted on carbon-coated copper grids, and stained in uranyl acetate in 50 % ethanol, followed by lead citrate. The stained sections were then coated with a thin layer of carbon, and fixed to coverslips with double-sided tape. The grids were coated with an approximate monolayer of diluted Ilford L-4 emulsion, by passing the coverslip through a film formed at the top of a 20-ml specimen tube (a modification of the Caro & van Tubergen (1962) loop method). The coverslips were attached to glass slides by double-sided tape, and the grids exposed at 5 C for 615 weeks in light-tight boxes containing silica gel. Development was carried out with Microdol-X for 3 min or with the ' compact' development mixture of Lettr6 & Paweletz (1966), for 1 min; in both cases at approximately 20 C. The 'compact' developer mixture (containing Phenidone as the developing agent) gave backgrounds too high for routine use with chloroplasts, which in general have a low level of labelling. Grain locations over chloroplasts were determined using Microdol-X development. Photographs of 131 chloroplasts from random locations in background-free areas were obtained by 2 different operators. We used the central region of the filamentous grain as the estimated Location of spinach choroplast DNA position of the original latent image (Caro & van Tubergen, 1962). We also scored photographs as to whether the grains were touching different membranes, as done by Comings & Okada (1973), in their nuclear DNA replication studies. [3H]thymidine incorporation into isolated chloroplast fractions Five-day-old light-grown disks (30 per plate) were incubated 24 h in 3H-TdR as in the autoradiography studies, and usually 180 with a total fresh weight of about 4 g used for chlorQplast isolation. Chloroplasts were obtained by a razor chopping method designed to give a high proportion of intact chloroplasts free of contaminating nuclei (Spencer & Wildman, 1964). Disks were chopped in a medium containing 0-25 M sucrose, 25 % Ficoll, 5 % dextran, 25 mM Tris-HCl pH 78, 1 /ig/ml Cleland's reagent, 2 mM CaCl3) 1 mM magnesium acetate, o-i % bovine serum albumin and 05 mg/ml cold thymidine (Honda, Hongladarom & Laties, 1966). The (...truncated)