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1.
A procedure is described for isolating photosynthetically active rhodoplasts (“red algal chloroplasts”) from the marine alga Griffithsia monilis. The rhodoplasts exhibited rates of CO 2 fixation and CO 2-dependent O 2 evolution in the order of 200 micromoles per milligram chlorophyll a per hour when illuminated with red or green light and were approximately 80% intact. The response of the rate of photosynthesis to the inorganic phosphate and pyrophosphate concentrations in the medium was qualitatively similar to that previously reported for spinach chloroplasts. Osmotically shocked rhodoplasts evolved O 2 from ferricyanide in red, but not in green, light and were completely uncoupled. Rhodoplast envelope rupture appeared to be accompanied by phycobilisome loss from the thylakoids. 相似文献
2.
Chloroplast suspensions from spinach ( Spinacia oleracea L.) were clearly resolved into intact and stripped chloroplasts by isopycnic centrifugation in density gradients of silica sol (“Ludox”) and polyethlene glycol. The intact chloroplasts fixed CO 2 and evolved O 2 more rapidly than the crude suspensions; the stripped chloroplasts were inactive. During the photosynthetic fixation of 14CO 2 in the intact chloroplasts recovered from the gradient, the 14C label was observed to spread through the photosynthetic intermediate pools, as well as into starch, which indicates that the purified chloroplasts are metabolically competent. This appears to be the first report of the retention of photosynthetic activity following the purification of chloroplasts in density gradients. 相似文献
3.
Application of water stress to isolated spinach ( Spinacia oleracea) chloroplasts by redutcion of the osmotic potentials of CO 2 fixation media below −6 to −8 bars resulted in decreased rates of fixation regardless of solute composition. A decrease in CO 2 fixation rate of isolated chloroplasts was also found when leaves were dehydrated in air prior to chloroplast isolation. An inverse response of CO 2 fixation to osmotic potential of the fixation medium was found with chloroplasts isolated from dehydrated leaves—namely, fixation rate was inhibited at −8 bars, compared with −16 or −24 bars. 相似文献
4.
Bicarbonate uptake by isolated chloroplast envelope membranes and intact chloroplasts of spinach ( Spinacia oleracea L. var. Viroflay) in darkness exhibited a similar dependency upon temperature, pH, time, and concentrations of isolated or attached envelope membranes. This similarity in uptake properties demonstrates the usefulness of the envelope membranes for the study of chloroplast permeability. Maximal rates for dark HCO 3- uptake by isolated envelope membranes and intact chloroplasts were more than sufficient to account for the maximal rates of photosynthetic CO 2 fixation observed with intact chloroplasts. The active species involved in the uptake process was found to be HCO 3- and not CO 2. The significance of HCO 3- uptake and its relationship to carbonic anhydrase and ribulose diphosphate carboxylase is discussed. Conditions for maximal HCO 3- uptake in darkness by intact chloroplasts were found to be similar to those required for maximal photosynthetic CO 2 fixation, suggesting that HCO 3- uptake by the envelope membrane may regulate photosynthetic CO 2 fixation. 相似文献
5.
Intact chloroplasts were isolated from sugarbeet leaves by the mechanical disruption technique normally used for spinach. The chloroplast pellet contained a ring of white irregularly shaped crystals which were identified as calcium oxalate. The chloroplasts were greater than 90% intact yet good rates of CO 2 fixation were only obtained when inorganic pyrophosphate or 3-phosphoglycerate were added to the assay medium. Chloroplasts free of calcium oxalate were prepared by purification on a three step Percoll gradient. These purified chloroplasts were highly intact and showed high rates of CO 2 fixation without adding inorganic pyrophosphate or 3-phosphoglycerate. With optimal assay conditions (0.2 mM orthophosphate and pH 8.0) rates of 110–130 mole per milligram chlorophyll per hour were routinely obtained. It is concluded that intact chloroplasts capable of high rates of CO 2 fixation can be prepared from sugarbeet leaves using a simple three step Percoll gradient.Abbreviations BSA
bovine serum albumin
- Chl
chlorophyll
- Pi
inorganic orthophosphate
- PPi
inorganic pyrophosphate
- PGA
3-phosphoglycerate
- EDTA
ethylenediamine tetraacetic acid
- EGTA
ethyleneglycol-bis-(aminoethyl ether)
- N,N
tetraacetic acid 相似文献
6.
Chloroplasts isolated from synchronous cultures of the unicellular green alga Chlamydomonas reinhardii, SAG 11-32/b (−), fix CO 2 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. 相似文献
7.
Chloroplasts, isolated from protoplasts of the green alga, Chlorella ellipsoidea, were estimated to be 99% intact by the ferricyanide-reduction assay, and gave CO 2 and PGA-dependent rates of O 2 evolution of 64.5 to 150 micromoles per milligram of chlorophyll per hour, that is 30 to 70% of the photosynthetic activity of the parent cells. Intact chloroplasts showed no carbonic anhydrase activity, but it was detected in preparations of ruptured organelles. Rates of photosynthesis, measured in a closed system at pH 7.5, were twice the calculated rate of CO 2 supply from the uncatalyzed dehydration of HCO 3− indicating a direct uptake of bicarbonate by the intact chloroplasts. Mass spectrometric measurements of CO 2 depletion from the medium on the illumination of chloroplasts indicate the lack of an active CO 2 transport across the chloroplast envelope. 相似文献
8.
Intact chloroplasts were isolated from sugarbeet leaves by the mechanical disruption technique normally used for spinach. The chloroplast pellet contained a ring of white irregularly shaped crystals which were identified as calcium oxalate. The chloroplasts were greater than 90% intact yet good rates of CO 2 fixation were only obtained when inorganic pyrophosphate or 3-phosphoglycerate were added to the assay medium. Chloroplasts free of calcium oxalate were prepared by purification on a three step Percoll gradient. These purified chloroplasts were highly intact and showed high rates of CO 2 fixation without adding inorganic pyrophosphate or 3-phosphoglycerate. With optimal assay conditions (0.2 mM orthophosphate and pH 8.0) rates of 110–130 μ mole per milligram chlorophyll per hour were routinely obtained. It is concluded that intact chloroplasts capable of high rates of CO 2 fixation can be prepared from sugarbeet leaves using a simple three step Percoll gradient. 相似文献
9.
Reduced osmotic potentials inhibited the rate of CO 2 fixation by isolated intact spinach ( Spinacia oleracea) chloroplasts. This inhibition was observed immediately after transfer of chloroplasts from a solution containing 0.33 m sorbitol to higher sorbitol concentrations, and the depressed rate remained constant. The inhibited CO 2 fixation could not be attributed to a decreased rate of photosynthetic electron transport, since NADP reduction was unaffected by subjecting the chloroplasts to low potentials. It could also not result from restricted permeability to CO 2, as CO 2 concentrations had no effect on the relative inhibition induced by the lowered potential. 相似文献
10.
This study examines the capacity of intact spinach ( Spinacia oleracea L.) chloroplasts to fix 14CO 2 when supplied with Benson-Calvin cycle intermediates in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Under these conditions, substantial 14CO 2 fixation occurred in the light but not in the dark when either dihydroxyacetone phosphate, ribulose 5-phosphate, fructose 6-phosphate, or fructose bisphosphate was added. The highest rate of 14CO 2 fixation (20-40 micromoles per milligram chlorophyll per hour) was obtained with dihydroxyacetone phosphate. In contrast, no 14CO 2 fixation occurred when 3-phosphoglycerate was used. 14CO 2 fixation in the presence of dihydroxyacetone phosphate and DCMU was inhibited by carbonylcyanide m-chlorophenylhydrazone, dl-glyceraldehyde, and pyridoxal 5′-phosphate. Low concentrations of O 2 (25-50 micromolar) stimulated 14CO 2 fixation, but the activity decreased with increasing O 2 concentrations. The fixation of 14CO 2 in the presence of DCMU and dihydroxyacetone phosphate was also observed in maize bundle sheath cells. These results provide direct evidence for cyclic photophosphorylation in intact chloroplasts. The activity measured is adequate to support all the extra ATP requirements for maximum rates of photosynthesis in these intact chloroplasts. 相似文献
11.
Summary The ultrastructure of chloroplasts from two genera of coenocytic green algae, Codium and Caulerpa, were examined after suspension in hypotonic solution and in detergent at various concentrations. The capacity of the suspensions to carry out CO 2-dependent and ferricyanide-dependent O 2 evolution was measured under the same conditions of osmotic strength and detergent concentration.The chloroplasts in the preparations were in the form of cytoplasts and gave rates of O 2 evolution comparable with those expected from undamaged chloroplasts. Suspension in hypotonic solution depressed the rate of CO 2-dependent O 2 evolution in both species, but this was partially restored in the Codium chloroplasts when these were re-suspended in iso-osmotic solutions. Major structural changes were observed only after suspension in buffer when the Codium chloroplasts lost their outer envelope, most of their stroma, and the thylakoids became swollen. Caulerpa chloroplasts were more variable in their response and, even when suspended in buffer only, the proportion of the plastids which had lost all of their stroma and thylakoid swelling was never as common as in Codium chloroplasts. However, once suspended in hyper-osmotic medium below 700 mosmolar, Caulerpa chloroplasts could not regain their capacity for CO 2-dependent O 2 evolution.Detergent treatment removed the cytoplast membrane but not the cytoplasmic material adhering to the chloroplast envelope. High concentrations of detergent were needed to cause loss of the chloroplast envelope, loss of stromal contents and unstacking of the thylakoids. Caulerpa chloroplasts were less sensitive to detergent than those of Codium. There was no indication that specific structures such as the thylakoid organizing body were resistant to detergent action. The results show that exposure to hypotonic solutions and to detergent results in less damage to these chloroplasts than it would to those of higher plants. It is proposed that the basis of this unusual resistance is not due to the properties of the chloroplast membranes but to the presence of material which coats the organelles during isolation. This material is likely to be identical with the sulphated xylo-mannogalactan isolated from the vacuole contents of these algae and which has the visco-elastic properties essential to allow the organelles to resist disruption by osmotic forces and disintegration by detergents. 相似文献
12.
Factors affecting CO 2 fixation in the spinach ( Spinacia oleracea) chloroplast were investigated. Free magnesium ions are shown to be highly inhibitory for photosynthetic CO 2 fixation in isolated intact spinach chloroplasts. The pH optimum for CO 2 fixation is about 8.5 but is dependent upon the reaction medium. Conditions are defined under which chloroplasts illuminated in the absence of CO 2 accumulate ribulose 1,5-diphosphate, and fix CO 2 in a subsequent dark period when high magnesium ion concentrations are provided. The regulation of photosynthetic CO 2 assimilation by these factors is discussed. 相似文献
13.
A system has been developed for the isolation of photosynthetically active chloroplasts from leaves of Populus deltoides. A high proportion of the chloroplasts appeared intact. The maximum rates of different photosynthetic processes were as follows: CO 2 fixation 3.5 micromoles per milligram chlorophyll per hour, noncyclic ATP synthesis 10 micromoles per milligram chlorophyll per hour, and cyclic ATP synthesis 300 micromoles per milligram chlorophyll per hour. 相似文献
14.
Chloroplasts from the cell wall mutant cw-15-2 of Chlamydomonas reinhardii were isolated by disruption of the cells in the Yeda press and fractionation through step gradients of Percoll. The resulting chloroplast fraction contained 80–85% intact chloroplasts. Electron micrographs of thin sections of the chloroplast fraction showed some cytoplasmic impurities, although almost no cytoplasmic ribosomes were detected by analysis of the ribosomal subunits.The isolated chloroplasts are active in photosynthetic O 2-evolution and CO 2-fixation, with the highest rates obtained in the presence of ATP.The chloroplast fraction also showed high rates of light-dependent in organello protein synthesis, with labelling of discrete chloroplast proteins known to be synthesized in the chloroplasts. 相似文献
15.
Effects of sulfur on photosynthesis in sugar beets ( Beta vulgaris L. cv. F58-554H1) were studied by inducing sulfur deficiency and determining changes in the photosynthesis of whole attached leaves and of isolated chloroplasts. The rates of photosynthetic CO 2 uptake by intact leaves, photoreduction of ferricyanide, cyclic and noncyclic photophosphorylation of isolated chloroplasts, and the rate of CO 2 assimilation by ribulose diphosphate carboxylase, decreased with decrease in total leaf sulfur from 2500 to about 500 μg g −1 dry weight. Sulfur deficiency reduced photosynthesis through an effect on chlorophyll content, which decreased linearly with leaf sulfur, and by decreasing the rate of photosynthesis per unit chlorophyll. There was only a small effect of sulfur deficiency on stomatal diffusion resistance to CO 2 until leaf sulfur decreased below 1000 μg g −1 when stomatal resistance became a more significant proportion of the total diffusion resistance to CO 2. Light respiration rates were positively correlated with photosynthesis rates and dark respiration was unchanged as leaf sulfur concentrations declined. 相似文献
16.
Photosynthesis, stroma-pH, and internal K + and Cl − concentrations of isolated intact chloroplasts from Spinacia oleracea, as well as ion (K +, H +, Cl −) movements across the envelope, were measured over a wide range of external KCl concentrations (1-100 millimolar). Isolated intact chloroplasts are a Donnan system which accumulates cations (K+ or added Tetraphenylphosphonium+) and excludes anions (Cl−) at low ionic strength of the medium. The internally negative dark potential becomes still more negative in the light as estimated by Tetraphenylphosphonium+ distribution. At 100 millimolar external KCl, potentials both in the light and in the dark and also the light-induced uptake of K+ or Na+ and the release of protons all become very small. Light-induced K+ uptake is not abolished by valinomycin suggesting that the K+ uptake is not primarily active. Intact chloroplasts contain higher K+ concentrations (112-157 millimolar) than chloroplasts isolated in standard media. Photosynthetic activity of intact chloroplasts is higher at 100 millimolar external KCl than at 5 to 25 millimolar. The pH optimum of CO2 fixation at high K+ concentrations is broadened towards low pH values. This can be correlated with the observation that high external KCl concentrations at a constant pH of the suspending medium produce an increase of stroma-pH both in the light and in the dark. These results demonstrate a requirement of high external concentrations of monovalent cations for CO2 fixation in intact chloroplasts. 相似文献
17.
Two green macroalgae, Codium decorticatum and Udotea flabellum, differ photosynthetically. Codium had high O 2-sensitive, and Udotea low O 2-insensitive, CO 2 compensation points; Codium showed a Warburg effect at seawater dissolved inorganic carbon levels and had photorespiratory CO 2 release, whereas Udotea did not. Seawater dissolved inorganic carbon levels did not saturate photosynthesis. For Codium, but not Udotea, the Warburg effect was increased by ethoxyzolamide, a carbonic anhydrase inhibitor, at high but not low pH. Isolated chloroplasts from both macroalgae showed a Warburg effect that was ethoxyzolamide-insensitive. In both macroalgae, chloroplastic and extrachloroplastic carbonic anhydrase activity was present. P-enolpyruvate carboxykinase (PEPCK) carboxylating activity in Udotea extracts was equivalent to that of ribulose bisphosphate carboxylase, and enzyme activities for C 4 acid metabolism and P-enolpyruvate regeneration were sufficient to operate a limited C 4-like system. In Udotea, malate and aspartate were early-labeled photosynthetic products that turned over within 60 seconds. Photorespiratory compounds were much less labeled in Udotea. Low dark fixation rates ruled out Crassulacean acid metabolism. A limited C 4-like system, based on PEPCK, is hypothesized to be the mechanism reducing photorespiration in Udotea. Codium showed no evidence of photosynthetic C 4 acid metabolism. Marine macroalgae, like terrestrial angiosperms, seem to have diverse photosynthetic modes. 相似文献
18.
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 CO 2 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 CO 2 a fixation and easily transported NADP, ferricyanide and ADP into the chloroplast. 相似文献
19.
Young expanding spinach leaves exposed to 14CO 2 under physiological conditions for up to 20 minutes assimilated CO 2 into lipids at a mean rate of 7.6 micromoles per milligram chlorophyll per hour following a lag period of 5 minutes. Label entered into all parts of the lipid molecule and only 28% of the 14C fixed into lipids was found in the fatty acid moieties, i.e. fatty acids were synthesized from CO 2in vivo at a mean rate of 2.1 micromoles per milligram chlorophyll per hour. Intact spinach chloroplasts isolated from these leaves incorporated H 14CO 3 into fatty acids at a maximal rate of 0.6 micromole per milligram chlorophyll per hour, but were unable to synthesize either the polar moieties of their lipids or polyunsaturated fatty acids. Since isolated chloroplasts will only synthesize fatty acids at rates similar to the one obtained with intact leaves in vivo if acetate is used as a precursor, it is suggested that acetate derived from leaf mitochondria is the physiological fatty acid precursor. 相似文献
20.
In Spinacia oleracea the kinetics of CO 2 fixation, of starch formation, and of changes in the levels of metabolites in chloroplasts and the surrounding medium has been investigated during light-dark and dark-light transitions with isolated intact chloroplasts. 相似文献
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