Evidence that the amount of chloroplast DNA exceeds that of nuclear DNA in mature leaves.

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intact chloroplasts and nuclei were allowed to settle for up to 1 h before the top 2 ml of the 5-ml homogenate was withdrawn.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.

Functional chloroplast polyribosomes from tobacco leaves.

Incubation of isolated to bacco chloroplasts with ingredients re quired for protein synthesis resulted in liberation of 70S ribosomnes and poly ribosomes that no longer sedimented with the chloroplasts. With increasing time of incubation, polyribosomes broke down to 70S monosomes. Similarly, microgram quantities of ribonuclease caused chloroplast polyribosomes to break down into monosomes. Both polyribosomes and 70S ribosomes that were isolated on sucrose density gra dients and tested separately in cell-free systems were capable of protein syn thesis; however, polyribosomes formed more protein per unit of RNA than monosomes did.

Chloroplast DNA from tobacco leaves.

DNA from tobacco leaf chloroplasts was isolated as a single component with a buoyant density in CsCl of 1.702 compared to 1.697 for nuclear DNA. 5-Methylcytosine is present in nuclear DNA but absent in chloroplast DNA. Chloroplast DNA, with a guanine-cytosine content of 43 percent, has a melting temperature of 86 degrees C and renatures completely on slow cooling, whereas nuclear DNA ( melting temperature, 84 degrees C; guanine-cytosine content, 40 percent) does not renature. About 9 percent of the total DNA in tobacco leaves is chloroplast DNA representing about 4.7 xX 10-(15) gram of DNA per chloroplast with a molecular weight of approximately 4 xX 10(7).

Crystalline fraction I protein: preparation in large yield.

Abolut 1 milligram of twice-recrystallized fraction I protein of constant specific ribulose diphosphate carboxylase activity per gram of leaves (fresh weight) has been obtained from each of seven different species of Nicotiana and 14 reciprocal, interspecific F(1) hybrids. Crystals are produced from honmogenates that have only been centrifuged to remove particulate matter.

Polypeptide composition of fraction 1 protein from parasexual hybrid plants in the genus Nicotiana.

Analysis of the subunit polypeptide composition of Fraction 1 proteins gives information on the expression of both nuclear and chloroplast genomes; the large subunits of the protein are coded by chloroplast DNA, whereas the small subunits are coded by nuclear DNA. Fraction 1 protein isolated from the leaves of parasexual hybrid plants derived from the fusion of protoplasts of Nicotiana glauca and N. langsdorffii contains the small subunit polypeptides of both parent species and the large subunit polypeptides of only N. glauca. Fraction 1 protein isolated from the leaves of a hybrid plant obtained after the uptake of chloroplasts of N. suaveolens by protoplasts of white tissue of a variegating mutant of N. tabacum contains the large subunit polypeptides of both N. suaveolens and N. tabacum, as well as the small subunit polypeptides of both these species.

Nuclear DNA codes for Nicotiana ferredoxin.

Ferredoxin was purified from 10 species of Nicotiana and spinach leaves. Fingerprints showed all to contain five major tryptic peptides. Some of the spinach peptides were different in RF and mobility from the Nicotiana peptides, but none of the Nicotiana ferredoxins had peptides which could distinguish one species of ferredoxin from another. Electrofocusing S-carbaminomethylcysteinyl ferredoxins showed spinach ferredoxin to have a more acidic and N. glutinosa ferredoxin a slightly more acidic isoelectric point than the other 9 Nicotiana species which were alike. Electro-focusing ferredoxin from the hybrid N. glutinosa female times N. glauca male resolved two bands or isozymes of ferredoxin, one corresponding to N. glutinosa, the other to N. glauca, the code for the latter having come from the DNA in the N. glauca pollen used to form the hybrid plant. N. glutinosa ferredoxin does not contain methionine and is different from N. tabacum and N. glauca ferredoxins which contain methionine. The N. glutinosa female times N. glauca male ferredoxin contained one-half the methionine found in N. glauca ferredoxin, thus confirming that some of the genetic information for ferredoxin in the hybrid was originally contained in the nuclear DNA of N. glauca.

Kinetic analysis of fraction I protein biosynthesis in young protoplasts of tobacco leaves.

At maximum inhibition chloramphenicol reduced [35S] methionine incorporation into acid-insoluble materials of sterile protoplasts from young tobacco leaves 5-7 cm in length by 30% compared to 70% by cycloheximide, indicating that 30% of the [35S] methionine became incorporated into chloroplast proteins and 70% into cytoplasmic proteins. [35S] Methionine became incorporated into both the large and small subunits of Fraction I protein, the major soluble protein of chloroplasts. Rifampicin and streptolydigin inhibited [3H] uridine incorporation into the 23 and 16 S rRNAs of chloroplasts to a much greater extent than into the 25 and 18 S cytoplasmic rRNAs. Rifampicin inhibited [35S] metionine incorporation into Fraction I protein after the third hour of incubation; streptolydigin after 2 h, the former evidently preventing initiation of mRNA for the large subunit of Fraction I protein and the latter its elongation. About 2.5 h was required between initiation of the large subunit mRNA synthesis, and appearance of the protein. It was estimated that 45 min is required for transcription of the mRNA which has a half-life of 2 h and that 105 min is required for its translation into approximately 350 amino acids constituting the large subunit monomeric polypeptide. The effect of chloramphenicol, cycloheximide and 2-(4-methyl-2,6-dinitroanaline)-N-methyl propionamide, the latter an inhibitor of protein initiation by 80 S ribosomes, on kinetics of Fraction I protein synthesis indicate that protoplasts contain a pool of small subunit polypeptides and that 30 min is required to polymerize the approximately 100 amino acids constituting the primary structure.

Chloroplasts in Living Cells and the String-of-Grana Concept of Chloroplast Structure Revisited.

In 1980, Wildman et al. (Bot Gaz 141: 24-36) proposed a three-dimensional model for chloroplast structure whereby the grana were arranged in non-overlapping rows, like beads on a string. This string-of-grana model was developed from phase microscope analysis of living cells and partially disrupted, isolated chloroplasts. However, models based on analyses by various electron microscope (EM) techniques (which inevitably encompass a relatively small fraction of the whole chloroplast) indicated that grana are interconnected in all directions by intergranal lamellae and not just along a single 'string.' Hence the string-of-grana model was not widely accepted. Recently, confocal laser scanning microscopy (CLSM) of both living and fixed cells, which gives views of the three-dimensional disposition of grana by imaging Photosystem II fluorescence over much larger sample volumes, that is, the entire chloroplast, has revealed that, although many grana are apparently not in any discernible arrangement, some are indeed present in strings of varying lengths in a range of taxa. The topic therefore warrants revisiting, using techniques, for example, such as EM tomography to assess the degree of variation in the geometry of intergranal connections in whole chloroplasts, and its possible functional consequences and developmental origins.