Chloroplasts as functional organelles in animal tissues

The marine gastropod molluscs Tridachia crispata, Tridachiella diomedea, and Placobranchus ianthobapsus (Sacoglossa, Opisthobranchia) possess free functional chloroplasts within the cells of the digestive diverticula, as determined by observations on ultrastructure, pigment analyses, and experiments on photosynthetic capacity. In the light, the chloroplasts incorporate H¹⁴CO₃^- in situ. Reduced radiocarbon is translocated to various chloroplast-free tissues in the animals. The slugs feed on siphonaceous algae from which the chloroplasts are derived. Pigments from the slugs and from known siphonaceous algae, when separated chromatographically and compared, showed similar components. Absorption spectra of extracts of slugs and algae were very similar. The larvae of the slugs are pigment-free up to the post-veliger stage, suggesting that chloroplasts are acquired de novo. with each new generation.     

Light-Dark Regulation of Starch Metabolism in Chloroplasts: II. Effect of Chloroplastic Metabolite Levels on the Formation of ADP-Glucose by Chloroplast Extracts

The rate of ADP-glucose formation from [¹⁴C]glucose 6-phosphate and ATP by the soluble fraction of lysed chloroplasts is studied as a function of the levels of metabolites (3-phosphoglycerate, orthophosphate, hexose monophosphate, and ATP) as determined in whole chloroplasts of Spinacia oleracea in light and dark.     

Partial Purification of Intact Chloroplasts from Chlamydomonas reinhardtii

Partially purified intact chloroplasts were prepared from batch cultures of both wild type (Wt) and a mutant strain of Chlamydomonas reinhardtii. Protoplasts were generated from log phase cultures of Wt (137c) and the phosphoribulokinase-deficient mutant F60 by incubation of the cells in autolysine. These protoplasts were suspended in an osmoticum, cooled, and then subjected to a 40 pounds per square inch pressure shock using a Yeda pressure bomb. The resulting preparation was fractionated on a Percoll step gradient which separated the intact chloroplasts from both broken chloroplasts and protoplasts.

The chloroplast preparation was not significantly contaminated with the cytoplasmic enzyme activity phosphoenolpyruvate carboxylase (>5%), and contained (100%) stromal enzyme activity ribulose-1,5-bisphosphate carboxylase. The chloroplast preparation is significantly contaminated by mitochondria, as determined by succinate dehydrogenase activity. Chloroplasts prepared from Wt cells retained CO₂-dependent O₂ photoevolution at rates in excess of 60 micromoles per milligram chlorophyll per hour, an activity which is severely inhibited by the addition of 10 millimolar KH₂PO₄. The chloroplasts are osmotically sensitive as determined by ferricyanide-dependent O₂ photoevolution.

     

Differences in Lipid Composition between Undifferentiated and Mature Maize Chloroplasts

Lipid compositions of undifferentiated maize (Zea mays) chloroplasts, capable of fixing CO₂, were compared with the lipid compositions of mature chloroplasts, which do not fix CO₂, located in both the mesophyll and bundle sheath cells. The major lipids found in all three chloroplast types were the glycolipids, monogalactosyl diglyceride and digalactosyl diglyceride, followed by decreasing amounts of sulfolipid, phosphatidyl glycerol, phosphatidyl choline, phosphatidyl inositol, and diphosphatidyl glycerol. Quantitative differences in lipid components were observed among the chloroplast types. The mesophyll and bundle sheath maize chloroplasts differed in their chlorophyll a/chlorophyll b ratios (2.27 and 4.13 respectively) and their content of glycolipid relative to chlorophyll (51.8% glycolipid to 20.9% chlorophyll and 84.5% glycolipid to 10.1% chlorophyll respectively). A comparison between the lipid compositions of maize mesophyll chloroplasts and mesophyll chloroplasts obtained from spinach, sugar beet, and tobacco showed many similarities.     

Simplified Procedure for the Isolation of Intact Chloroplasts from Chlamydomonas reinhardtii

A simple procedure that yields highly purified intact chloroplasts from Chlamydomonas reinhardtii is described. This procedure involves breakage of cell wall-deficient cells by passing them through a narrow bore syringe needle. The intact chloroplasts are then purified from the crude homogenate by differential centrifugation and Percoll gradient centrifugation. This procedure generates relatively high yields of chloroplasts capable of CO₂ fixation. These chloroplasts were characterized by electron microscopy, marker enzyme analysis, and ferricyanide exclusion. Transmission electron microscopy indicates that these chloroplasts retain their pyrenoids and eyespots. Scanning electron microscopy confirms that the characteristic cup shape of C. reinhardtii chloroplasts persists in vitro. This rapid, inexpensive procedure produces chloroplasts that should be useful for researchers studying the biochemistry and cell biology of C. reinhardtii chloroplasts.     

Nitrite Reduction in Reconstituted and Whole Spinach Chloroplasts during Carbon Dioxide Reduction

Nitrite reduction in either whole, isolated spinach chloroplasts (Spinacia oleracea L.) or in reconstituted spinach chloroplasts is stimulated by a short period of photosynthetic CO₂ fixation in the light prior to nitrite addition. With reconstituted chloroplasts, a similar stimulation can be obtained in nitrite reduction without CO₂ fixation by the addition of dihydroxyacetone phosphate or fructose 6-phosphate. Specific intermediate metabolites of the photosynthetic carbon reduction cycle may have a regulatory role in nitrite reduction in chloroplasts in the light.     

Isolation of Intact Chloroplasts from Dunaliella tertiolecta

Cells of Dunaliella tertiolecta from the log phase of growth were broken by rapid extrusion at low pressure through a Yeda press and the chloroplasts were isolated by centrifugation through a Percoll gradient. Osmolarity of the growth media, the suspending media, and the Percoll gradient was kept identical to minimize change in chloroplast volume and mitochondrial entrapment. The isolated intact chloroplasts were obtained in a 30 to 50% yield based on chlorophyll and were stable to washing with buffered medium. Isolated chloroplast yield and purity was dependent on cell culture condition; a cycle of 16 hours light and 8 hours dark with continuous high CO₂ was optimum. Isolated chloroplasts were about 90% intact by microscopic examination, ferricyanide-dependent O₂ evolution, and the distribution of four stromal enzymes. Enzymes associated with glycolate metabolism were not in the chloroplast fraction. The isolated chloroplasts with 10 millimolar bicarbonate evolved 24 micromoles of O₂ and fixed 21 micromoles of CO₂ per hour per milligram of chlorophyll, which rates were about one-third of those by whole cells. The inhibition of oxygen evolution by 10 millimolar phosphate was reversed by P-glycerate. Whole chloroplasts were also isolated from cells adapted to low CO₂ in air for 24 hours. On low CO₂ the cells excreted more gelatinous material, which had to be removed with additional washing of the cells, before it was possible to obtain good chloroplast preparations.     

The Involvement of Aspartate and Glutamate in the Decarboxylation of Malate by Isolated Bundle Sheath Chloroplasts from Zea mays

Aspartate or glutamate stimulated the rate of light-dependent malate decarboxylation by isolated Zea mays bundle sheath chloroplasts. Stimulation involved a decrease in the apparent K_m (malate) and an increased maximum velocity of decarboxylation. In the presence of glutamate other dicarboxylates (succinate, fumarate) competitively inhibited malate decarboxylation by intact chloroplasts with respect to malate with an apparent K_i of about 6 millimolar. For comparison the K_i for inhibition of nicotinamide adenine dinucleotide phosphate-malic enzyme from freshly lysed chloroplasts by these dicarboxylates was 15 millimolar. A range of compounds structurally related to aspartate stimulated malate decarboxylation by intact chloroplasts. K_a values for stimulation at 5 millimolar malate were 1.7, 5, and 10 millimolar for l-glutamate, l-aspartate, and β-methyl-dl-aspartate, respectively. Certain compounds, notably cysteic acid, which stimulated malate decarboxylation by intact chloroplasts inhibited malate decarboxylation by nicotinamide adenine dinucleotide phosphate-malic enzyme obtained from lysed chloroplasts and assayed under comparable conditions. It was concluded that aspartate, glutamate, and related compounds affect the transport of malate into the intact chloroplasts and that malate translocation does not take place on the general dicarboxylate translocator previously reported for higher plant chloroplasts.     

Uptake of l-Ascorbate by Intact Spinach Chloroplasts

Uptake of l-[1-¹⁴C]ascorbate by intact ascorbate-free spinach (Spinacia oleracea L. cv Vital^r) chloroplasts has been investigated using the technique of silicone oil filtering. Rates greater than 100 micromoles per milligram chlorophyll per hour (external concentration, 10 millimolar) of ascorbate transport were observed. Ascorbate uptake into the sorbitol-impermeable space (stroma) followed the Michaelis-Menten-type characteristic for substrate saturation. A K_m of 18 to 40 millimolar was determined. Transport of ascorbate across the chloroplast envelope resulted in an equilibrium of the ascorbate concentrations between stroma and medium. A pH optimum of 7.0 to 7.5 and the lack of alkalization of the medium upon ascorbate uptake suggest that only the monovalent ascorbate anion is able to cross the chloroplast envelope. The activation energy of ascorbate uptake was determined to be 65.8 kilojoules (16 kilocalories) per mole (8 to 20°C). Interference of ascorbate transport with substrates of the phosphate or dicarboxylate translocator could not be detected, but didehydroascorbate was a competitive inhibitor. Preloading of chloroplasts with didehydroascorbate resulted in an increase of V_max but did not change the K_m for ascorbate. Millimolar concentrations of the sulfhydryl reagent p-chloromercuriphenyl sulfonate inhibited ascorbate uptake. The data are interpreted in terms of ascorbate uptake into chloroplasts by the mechanism of facilitated diffusion mediated by a specific translocator.     

Photosynthetic Properties of Chloroplasts from Chlamydomonas reinhardii

Chloroplasts isolated from synchronous cultures of the unicellular green alga Chlamydomonas reinhardii, SAG 11-32/b (−), fix CO₂ at rates between 25 and 50 micromoles per milligram chlorophyll per hour. The upper value is approximately half of the rate of the intact cell.

During storage in the dark on ice, the chloroplast preparation loses 30 to 50% of its CO₂ fixing capability per hour. Under reducing conditions (+ 1 millimolar dithiothreitol), this loss of activity is about twice as fast. The same reducing conditions stimulate CO₂ fixation in the light.

High concentrations of inorganic phosphate (>2 millimolar) inhibit CO₂ fixation. This inhibition is overcome by the addition of glycerate 3-phosphate. It is concluded that chloroplasts from C. reinhardii possess a higher plant type phosphate translocator. With respect to dependency upon light intensity, pH and Mg²⁺ concentration, the results were similar to that reported for chloroplasts from higher plants. However, in contrast to higher plant chloroplasts, maximum CO₂ fixation is observed at the relatively low osmotic concentration of 0.12 molar mannitol in the reaction buffer.

     

Hydrogenase-Mediated Activities in Isolated Chloroplasts of Chlamydomonas reinhardii

Isolated intact chloroplasts of Chlamydomonas reinhardii were found to catalyze photoreduction of CO₂ in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea when adapted under an atmosphere of H₂ demonstrating the association of a hydrogenase and anaerobic adaptation system with these plastids. The specific activity of photoreduction was approximately one third that detected in cells and protoplasts. Photoreduction was found to have a lower osmoticum optimum relative to aerobically maintained chloroplasts (50 millimolar versus 120 millimolar mannitol). 3-Phosphoglycerate (3-PGA) stimulated photoreduction up to a peak at 0.25 millimolar beyond which inhibition was observed. In the absence of 3-PGA, inorganic phosphate had no effect on photoreduction but in the presence of 3-PGA, inorganic phosphate also stimulated the reaction. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone inhibited photoreduction but inhibition by the former could be partially overcome by exogenously added ATP. The intact plastid can also catalyze photoevolution of H₂ while lysed chloroplast extracts catalyzed the reduction of methyl viologen by H₂. Both reactions occurred at rates approximately one-third of those found in cells. The oxyhydrogen reaction in the presence or absence of CO₂ was not detected.     

Activation and Deactivation of H-ATPase in Intact Chloroplasts

The light activation mechanism of the latent H⁺-ATPase was investigated in intact spinach (Spinacia oleracea, Hybrid 424) chloroplasts. The following observations were made. (a) Photosystem I electron acceptors such as methyl viologen, nitrite, oxaloacetate, etc., inhibit the light activation of the enzyme. (b) The electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) fully inhibits the process. (c) Ascorbate plus diaminodurene or dithionite can restore light activation in DCMU-poisoned chloroplasts. (d) The activated state of the enzyme decays rather slowly (within a few minutes) after illumination of the intact chloroplasts. (e) The rate of dark decay is accelerated by oxidants (H₂O₂ or ferricyanide) and slowed down by dithiothreitol.

It is suggested that the physiological mechanism for regulation of the H⁺-ATPase involves oxidation and reduction reactions in a manner which resembles the regulation of the light-activated carbon cycle enzymes.

     

Nomenclature for Isolated Chloroplasts

THE nomenclature used to describe the “intactness” or degree of breakage of isolated chloioplasts has been most confusing in the past few years. This resulted chiefly from reports that chloroplasts should be isolated carefully and rapidly to obtain high rates of CO₂ fixation (50–250 µmol/mg chlorophyll/h). This type of chloroplast cannot translocate NADP, ferricyanide or ADP through its intact limiting membrane or envelope. “Class I” chloroplasts, as seen by phase contrast microscopy, had previously been considered to be similar to the in vivo situation. Unfortunately, in vitro they showed only low rates of CO₂ a fixation and easily transported NADP, ferricyanide and ADP into the chloroplast.     

Photosynthetic Reactions in the Marine Alga Codium vermilara: I. CO(2) Fixation and Hill Reaction in Isolated Chloroplasts

Chloroplasts were isolated from the marine alga Codium vermilara (Siphonales). The isolated chloroplasts were active in CO₂ fixation in the light at a rate comparable to the rates obtained by fragments of thalli. Maximal rates of CO₂ fixation by isolated chloroplasts from Codium were obtained in the presence of salt or sorbitol isoosmotic with sea water. The conditions of isolation of Codium chloroplasts are much less stringent than those required for active chloroplasts from higher plants. The isolated chloroplasts comprise a homogeneous population of the intact “class I” type, as based on microscopic observations and on their inability to reduce ferricyanide unless osmotically shocked. The intact chloroplasts are able to reduce p-benzoquinone at a high rate.     

A Lipid Requirement for Photosystem I Activity in Heptane-extracted Spinach Chloroplasts

A lipid requirement for photosystem I activity in Spinacia oleracea chloroplasts has been characterized. The transfer of electrons from tetramethyl-p-phenylenediamine through the chloroplast photosystem to viologen dye was used as an assay of photosystem I activity. Activity is diminished by prolonged heptane extraction and is partially restored by readdition of the extracted lipid. Extracted chloroplasts require plastocyanin for maximal restoration of activity. The effect of lipid extract in restoration is partially replaced by triglycerides containing unsaturated, C₁₈ fatty acids. Various potential redox carriers which occur naturally in chloroplasts do not substitute for extracted lipid. Galacto-lipids, sulfolipids, and phospholipids are not involved in the restoration of activity.     

Zinc-inhibited Electron Transport of Photosynthesis in Isolated Barley Chloroplasts

In isolated barley chloroplasts, the presence of 2 millimolar ZnSO₄ inhibits the electron transport activity of photosystem II, as measured by photoreduction of dichlorophenolindophenol, O₂ evolution, and chlorophyll a fluorescence. The inhibition of photosystem II activity can be restored by the addition of the electron donor hydroxylamine or diphenylcarbazide, but not by benzidine and MnCl₂. These observations suggest that Zn inhibits electron flow at the oxidizing side of photosystem II at a site prior to the electron donating site(s) of hydroxylamine and diphenylcarbazide. No inhibition of photosystem I-dependent electron transport by 3 millimolar ZnSO₄ is observed. However, with concentrations of ZnSO₄ above 5 millimolar, photosystem I activity is partially inactivated. Washing Zn²⁺-treated chloroplasts partially restores the O₂-evolving activity.     

Distribution of Protein-bound Hexosamine in Chloroplasts

Intact chloroplasts of spinach (Spinacia oleracea L.), sunflower (Helianthus annuus L.), and maize (Zea mays L.) mesophyll cells contained 0.33, 0.50, and 0.14% of bound hexosamine on a protein basis, respectively. Undifferentiated maize chloroplasts contained 0.19%. Values for chloroplast lamellae were, respectively, 0.16, 0.18, 0.12, and 0.06% and for envelope membranes they were 1.6, 2.5, 3.8, and 2.7%. Thus most of the hexosamine of chloroplasts is located in the envelope membrane.     

Redox Reactions between Kaempferol and Illuminated Chloroplasts

Bleaching of kaempferol by illuminated chloroplasts was observed at 380 nanometers. The photobleaching was stimulated by methyl viologen and suppressed by superoxide dismutase indicating the participation of O₂⁻ in the reaction. An electron transfer inhibitor on the oxidizing side of photosystem II, carbonylcyanide m-chlorophenylhydrazone (CCCP), stimulated the photobleaching and 3-(3,4-dichlorophenyl)-1,1-dimethylurea partially suppressed it. The stimulation by CCCP suggests that kaempferol is also bleached on the oxidizing side of photosystem II. The spectrum of kaempferol bleaching in the presence of methyl viologen was the same as that in the presence of CCCP having a maximum in absorbance decrease at around 380 nanometers. When kaempferol was oxidized by KMnO₂ or KO₂, the oxidized minus reduced difference spectra had also a negative peak at about 380 nanometers. The results suggest that kaempferol was oxidized by illuminated chloroplasts.

The rate of kaempferol photooxidation increased as its concentration was increased from 1 to 100 micromolar. The rate of quercetin photooxidation also increased as its concentration was increased from 1 to 100 micromolar. Concentration of quercetin glycosides higher than 10 micromolar was required to detect their photobleaching by illuminated chloroplasts. From these results, it is postulated that flavonols function as antioxidants in chloroplasts.

     

Involvement of Singlet Oxygen in 5-Aminolevulinic Acid-Induced Photodynamic Damage of Cucumber (Cucumis sativus L.) Chloroplasts

Cucumber (Cucumis sativus L., cv Poinsette) plants were sprayed with 20 millimolar 5-aminolevulinic acid and then incubated in the dark for 14 hours. The intact chloroplasts were isolated from the above plants in the dark and were exposed to weak light (250 micromoles per square meter per second). Within 30 minutes, photosystem II activity was reduced by 50%. The singlet oxygen (¹O₂) scavengers, histidine and sodium azide (NaN₃) significantly protected against the damage caused to photosystem II. The hydroxyl radical scavenger formate failed to protect the thylakoid membranes. The production of ¹O₂ monitored as N,N-dimethyl p-nitrosoaniline bleaching increased as a function of light exposure time of treated chloroplasts and was abolished by the ¹O₂ quencher, NaN₃. Membrane lipid peroxidation monitored as malondialdehyde production was also significantly reduced when chloroplasts were illuminated in the presence of NaN₃ and histidine. Protochlorophyllide was the most abundant pigment accumulated in intact chloroplasts isolated from 5-aminolevulinic acid-treated plants and was probably acting as type II photosensitizer.     

Nucleic Acid Synthesis in the Chloroplasts of Acetabularia mediterranea

Radioautographic and radiochemical techniques were used to establish the presence of replicating DNA in the chloroplasts of Acetabularia mediterranea. These techniques also demonstrated that these chloroplasts synthesize RNA. It was found that label from thymine was also incorporated into DNA and RNA in these chloroplasts.

With the establishment of protein and nucleic acid synthesis in Acetabularia chloroplasts, it is clear that these chloroplasts carry out those metabolic processes which are most characteristic of autonomous cells.