Bean Chloroplasts Research Articles

Base composition of DNA from chloroplasts and nuclei of Phaseolus vulgaris.

WHEN the first reports of the isolation and biochemical study of chloroplast DNA from higher plants appeared in 1963, there was a discrepancy between the two sets of findings. J. T. O. K.1,2, using chemical analysis, reported that broad bean chloroplast DNA had a guanine-cytosine content (37.4 per cent GC) close to, but slightly (and significantly) lower than, that of the nuclear DNA (39.4 per cent GC). Chun, Vaughan and Rich3 using spinach and beet, and later other workers using swiss chard4, tobacco5 and other plants6, reported, however, that DNA from chloroplast preparations analysed by CsCl equilibrium density gradient centrifugation contained—in addition to a component of similar density to nuclear DNA—one or two components of much higher density (and therefore of much higher GC content): these dense components they considered to be the true chloroplast DNA. Recent and more rigorous studies7–9 have now cast considerable doubt on the identification of these high density bands with chloroplast DNA.

The distribution and stability of α-tocopherol in subcellular fractions of broad bean leaves

A method is described which enables the preparation of chloroplasts with the minimum enzymic breakdown of α-tocopherol. Using this method, it was shown that α-tocopherol is confined to the chloroplasts of young broad bean leaves. Fifty per cent of this α-tocopherol is removed from the chloroplast lamella by treatment with digitonin. This α-tocopherol is not located in plastoglobuli and is not removed from the lamellae by sonication.

Inhibition of Photosynthetic Electron Transport by Ioxynil

THE herbicide ioxynil (4-hydroxy-3,5-diiodo benzonitrile)1 has been found to inhibit the photoreduction of ferricyanide by chloroplasts from a range of plants2 and also NADP photoreduction, the accompanying photophosphorylation and carbon dioxide fixation by the broad bean (Vicia faba) chloroplasts3. Recently, Paton and Smith4 further reported that ioxynil inhibited the photoreduction of endogenous plastoquinone in broad bean chloroplasts, even in the presence of ascorbate and indophenol. It was previously found5 that although o-phenanthroline and the herbicide DCMU inhibit the photoreduction of endogenous plastoquinone they do not inhibit the enhanced photoreduction in the presence of ascorbate and indophenol. Furthermore, ascorbate and indophenol reduce plastoquinone in darkness6, in a reaction which has been shown to be non-enzymatic7. No other inhibitors previously investigated have inhibited the dark reduction and we therefore decided to investigate the effects of ioxynil on plastoquinone reduction in more detail.

The Ultrastructure ofPhaseolus vulgarisChloroplasts

A comparison of bean chloroplasts after being fixed in potassium perman ganate, osmium, and formaldehyde coupled with negative staining shows that the general organization of the chloroplast is similar in all cases. However, the mature chloroplasts of bean vary considerably in the extent and orientation of the internal membranes—the grana and the interconnecting membranes between the grana. The interconnecting membranes are thin, branching, flexuous structures. This is illustrated by serial sections and by crossand face-view sections of osmium fixed chloroplasts and the best model, which allows for a considerable flexibility in the orientation of the grana and also describes the thin interconnecting mem branes between the grana, is the grana-fretwork system proposed by Weier (1961). Furthermore, the comparative studies show that the internal regions of grana are separated from the stroma and that the grana-fretwork systems appear to be a continuous membrane system. This membrane is single along the frets, end compartments, and granal margins. It is double in the partitions of the grana but separated by a component, possibly a cementing material, which does not stain. It is suggested that this membrane is structurally similar in the chloro plasts of higher plants, but that its overall organization may vary from one plant to another.

Studies on RNA synthesis in chloroplast preparations

It has been found (Kirk, 1964) that highly purified preparations of broad bean chloroplasts incorporate radio-activity from 14C-ATP into a substance having the properties of RNA. The reaction is similar in certain respects (requirement for all four nucleoside triphosphates, sensitivity to deoxyribonuclease and actinomycin D) to the RNA polymerase enzymes that have been found in other organisms ( Weiss, 1960; Hurwitz et al. , 1960 ; Stevens, 1960; Huang et al. , 1960 ; Chamberlin and Berg, 1962). It was suggested that the activity is due to a chloroplast RNA polymerase, working with chloroplast DNA as a template. Evidence has now been obtained by other workers (Dr. S.G. Wildman, personal communication) for RNA synthesis in tobacco chloroplast preparations. In this paper, further studies on the enzyme are described, with particular reference to apparent differences between the reactions catalyzed by the chloroplast and nuclear fractions.

Studies on The Greening of Dark-Grown Bean Plants II. Development of Photochemical Activity

A study of the photochemical activity of plastids isolated from etiolated bean leaves which had been illuminated for increasing periods was undertaken. Proplastids isolated from etiolated leaves were inactive in the Hill reaction. Photochemical ferricyanide reduction was obtained with plastids isolated from etiolated leaves which had been illuminated for 6 hr. The reduction increased to a maximum value of 400 p.lloles ferricyanide/mg chlorophyll/hr after 10 hr of illumina� tion and thereafter decreased to 300,umoles ferricyanidejmg chlorophylljhr for mature bean chloroplasts. Plastids isolated from etiolated leaves illuminated for 16 hr reduced nicotinamide adenine dinucleotide phosphate (NADP) at a rate less than half that of mature chloroplasts, while B�hr plastids showed no NADP reduction.

The deoxyribonucleic acid of broad bean chloroplasts

1. 1. The question of whether higher-plant chloroplasts contain DNA has been investigated. 2. 2. A method of obtaining highly purified broad bean chloroplasts by high-speed density-gradient centrifugation is described. The chloroplasts thus obtained have a DNA content, as measured by the diphenylamine reaction, of about 0.15% of the dry weight. About one-third of this DNA may be removed by repeated density-gradient centrifugation. 3. 3. 66% of the diphenylamine-positive material from the chloroplasts may be recovered after deproteinization and alcohol precipitation. Of this, 93% is rendered soluble by DNAase (EC 3.1.4.5) treatment, indicating that it is DNA. 4. 4. The chloroplast DNA is much less readily extracted by HClO 4 than is nuclear DNA. Using gentle depurination followed by ion-exchange chromatography, the molar ration of adenine to guanine has been found to be 1.67 for chloroplast DNA and 1.54 for nuclear DNA: this difference, although small, is statistically significant at the 0.1% level. These results suggest that the DNA in the chloroplast preparations is not contaminating nuclear DNA.

The deoxyribonucleic acid of broad bean chloroplasts

The deoxyribonucleic acid of broad bean chloroplasts

The deoxyribonucleic acid of broad bean chloroplasts

1. The question of whether higher-plant chloroplasts contain DNA has been investigated. 2. A method of obtaining highly purified broad bean chloroplasts by high-speed density-gradient centrifugation is described. The chloroplasts thus obtained have a DNA content, as measured by the diphenylamine reaction, of about 0.15% of the dry weight. About one-third of this DNA may be removed by repeated density-gradient centrifugation. 3. 66% of the diphenylamine-positive material from the chloroplasts may be recovered after deproteinization and alcohol precipitation. Of this, 93% is rendered soluble by DNAase (EC 3.1.4.5) treatment, indicating that it is DNA. 4. The chloroplast DNA is much less readily extracted by HClO4 than is nuclear DNA. Using gentle depurination followed by ion-exchange chromatography, the molar ration of adenine to guanine has been found to be 1.67 for chloroplast DNA and 1.54 for nuclear DNA: this difference, although small, is statistically significant at the 0.1% level. These results suggest that the DNA in the chloroplast preparations is not contaminating nuclear DNA.

THE INTRACELLULAR DISTRIBUTION OF SOME METALLIC ELEMENTS IN LEAVES

Stocking, C. Ralph, and Alpaslan Ongun. (U. California, Davis.) The intracellular distribution of some metallic elements in leaves. Amer. Jour. Bot. 49(3): 284–289. Illus. 1962.—A comparison of the potassium, sodium, calcium, magnesium, and nitrogen levels of chloroplasts isolated in an aqueous sucrose medium and by the nonaqueous method reveals that large amounts of these elements are lost from the plastids during their isolation in sucrose solution. Nonaqueously isolated chloroplasts of bean and tobacco leaves contained over 40% of the total leaf potassium, magnesium, and calcium. High levels of nitrogen up to 70% of the total leaf nitrogen were found associated with nonaqueously isolated chloroplasts. As much as 40–50% of this nitrogen is lost when chloroplasts are isolated in a sucrose solution. Electron photomicrographs of nonaqueously isolated chloroplasts reveal only a partial disruption of the internal structure and a loss of the plastid membrane during isolation.

Fatty Acids of Spinach Chloroplasts

The photosynthetic apparatus of higher plants consists of discrete cytoplasmic organelles, the chloroplasts, which contain proteins, lipids, pigments, water, and other substances. Relatively little is known, apparently, concerning the composition of the lipid fraction of the chloroplast. Menke (8), studying chloroplasts of spinach, Spinacia oleracea, found that essentially all the lipids of the leaf were located in the chloroplasts. The chloroplasts had an average lipid content of 30.9 % of the dry weight, while the cytoplasm had a lipid content of only 0.5 %. For this reason, a number of earlier studies of the lipid content and composition of green leaves as a whole are pertinent to the present problem. Chibnall and Channon (3), studying the fatty acids of leaves of cabbage, Brassica oleracea, found that approximately 10.7 % of the fatty acid fraction consisted of saturated acids, and 89.3 % of unsaturated acids. The saturated fraction was composed of 70 % palmitic acid and 30 % stearic acid. The unsaturated fraction included large quantities of linoleic and linolenic acids. Speer et al. (13) examined the lipids of spinach leaves, finding that 53 % of the fatty acidls occurred in the free form, with 47 % as glycerides. The saturated acids consisted chiefly of palmitic and stearic acids. Oleic acid made up 26.3 %, linoleic acid 34.7 %, and linolenic acid 12.7 % of the unsaturated lipid fraction. The fatty acids of the forage grasses Dactylis glomierata and Lolium perenne were examined by Smith and Chibnall (12). Saturated acids composed 11 % of the total in Dactylis and 12 % in Lolium; the corresponding figures for oleic acid were 16 and 22 %, for linoleic acid 31 and 26 %, and for linolenic acid 42 and 40 %, respectively. According to Chibnall (2), cabbage leaves contain 17.5 % glycerides, 12.3 % waxes, 4.5 % sterols, and 18.4 % phospholipids. Comparable figures for cocksfoot grass, Dactylis glomnerata, were 38.0 % glycerides, 23.5 % waxes, 3.0 % sterols, and 1.5 % phospholipids. Menke (9) isolated chloroplasts from spinach leaves by centrifugation; he found that their lipid content was approximately 37 %. The ether-soluble fraction of the chloroplasts is a complex mixture of pigments, fatty acids, glycerides, phosphatides, and sterols. Bot (1) isolated grana from chloroplasts of Lathyrus odorattis and Spinacia oleracea. These were shown to have an ether-soluble fraction, excluding chlorophyll, of 18 to 30 % of the dry weight in sweet pea and 26 to 32 % in spinach. Comar (4) determined the lipid content of spinach chloroplasts as 34%. Menke and Jacob (10) extracted isolated chloroplasts of spinach with ether or ether-alcohol mixtures. The extracts were fractionated into a larger acetonesoluble fraction, making up about 80 % of the total, and containing the pigments, triglycerides and sterols, and an acetone-insoluble fraction including phosphatides and waxes. The pigment-free portion of the acetone-soluble fraction consisted of 5.1 % glycerol, 42.2 % fatty acids, 49.0 % triglycerides, and 2.1 % sterols. Sisakyan and Smirnov (11) found that fatty acids composed 57.8 % of the dry weight of the chloroplasts of sugar beet, 48.8 % in sunflower, and 60.0 % in red clover. Isolated chloroplasts of sunflower incorporated acetate-C'4 into higher fatty acids in either light or darkness, but bean chloroplasts did not. In manometric experiments, chloroplasts of bean oxidized palmitic, oleic, linoleic, and linolenic acids. Therefore, the chloroplasts are metabolically active in both fatty acid synthesis and oxidation. Zill and Harmon (14) investigated the chloroplast lipids of spinach by the technique of chromatography on silicic acid columns. Of the crude whole spinach extract 24 % was represented by two monoglycerides, which were not specifically identified. Further studies of chloroplast lipids by these workers are in progress (personal communication). Debuch (5) has recently analyzed the fatty acid composition of chloroplasts of Antirrhinum majus, a plastid mutant of this species, and Alliumn porrum by means of gas chromatography. C1l6 fatty acids composed 10 to 20 % and C18 fatty acids composed 80 to 90 % of the total. Linolenic acid was the component present in greatest amount (44.1-71.3 % of the total), linoleic acid (15.0-29.2 %) and palmitic acid (9.519.7 %) were the other principal constituents. The studies reported in this paper were designed to determine the fatty acid composition of chloroplasts obtained from spinach leaves.

The Hill Reaction of Red Kidney Bean Chloroplasts.

The Hill reaction rates of red kidney bean chloroplasts prepared by usual procedures were found to be low compared to those of spinach. Active chloroplasts could be prepared only if Versene was added to the homogenizing buffer. Raising the pH of the grinding buffer to S or over could substitute in part for the presence of Versene. Once the leaves were ground, no further changes in chloroplast activity could be induced by reciprocation of the above treatment. Chloroplasts with low activity have apparently been damaged in a dark step, not in the light-dependent part of the Hill reaction. 8 references, 3 figures, 5 tables.

PHOSPHORYLASE IN THE CHLOROPLASTS OF WHEAT

Phosphorylase activity was demonstrated in the chloroplasts of wheat, bean, and sunflower by incubating living starch-free sections with glucose-1-phosphate in acetate buffer at pH 5.8–6.0.