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1.
Adenylate concentrations were measured in intact chloroplasts under a variety of conditions. Energy charge was significant in the dark and increased in the light, but remained far below values expected from observed phosphorylation potentials in broken chloroplasts, which were 80 000 M ?1 or more in the light. With nitrite as electron acceptor, phosphorylation potentials in intact chloroplasts were about 80 M ?1 in the dark and only 300 M ?1 in the light. Similar phosphorylation potentials were observed, when oxaloacetate, phosphoglycerate or bicarbonate were used as substrates. ΔG′ ATP was ?42 kJ/mol in darkened intact chloroplasts, ?46 kJ/mol in illuminated intact chloroplasts and ?60 kJ/mol in illuminated broken chloroplasts. Uncoupling by NH 4Cl, which stimulated electron transport to nitrite or oxaloacetate and decreased the proton gradient, failed to decrease the phosphorylation potential of intact chloroplasts. Also, it did not increase the quantum requirement of CO 2 reduction. It is concluded that the proton motive force as conventionally measured and phosphorylation potentials are far from equilibrium in intact chloroplasts. The insensitivity of CO 2 reduction and of the phosphorylation potential to a decrease in the proton motive force suggests that intact chloroplasts are over-energized even under low intensity illumination. However, such a conclusion is at variance with available data on the magnitude of the proton motive force. 相似文献
2.
Cytoplasmic streaming occurs in most plant cells and is vitally important for large cells as a means of long-distance intracellular transport of metabolites and messengers. In internodal cells of characean algae, cyclosis participates in formation of light-dependent patterns of surface pH and photosynthetic activity, but lateral transport of regulatory metabolites has not been visualized yet. Hydrogen peroxide, being a signaling molecule and a stress factor, is known to accumulate under excessive irradiance. This study was aimed to examine whether H 2O 2 produced in chloroplasts under high light conditions is released into streaming fluid and transported downstream by cytoplasmic flow. To this end, internodes of Chara corallina were loaded with the fluorogenic probe dihydrodichlorofluorescein diacetate and illuminated locally by a narrow light beam through a thin optic fiber. Fluorescence of dihydrodichlorofluorescein (DCF), produced upon oxidation of the probe by H 2O 2, was measured within and around the illuminated cell region. In cells exhibiting active streaming, H 2O 2 first accumulated in the illuminated region and then entered into the streaming cytoplasm, giving rise to the expansion of DCF fluorescence downstream of the illuminated area. Inhibition of cyclosis by cytochalasin B prevented the spreading of DCF fluorescence along the internode. The results suggest that H 2O 2 released from chloroplasts under high light is transported along the cell with the cytoplasmic flow. It is proposed that the shift of cytoplasmic redox poise and light-induced elevation of cytoplasmic pH facilitate the opening of H +/OH ?-permeable channels in the plasma membrane. 相似文献
3.
Cytoplasmic streaming in plant cells is an effective means of intracellular transport. The cycling of ions and metabolites
between the cytosol and chloroplasts in illuminated cell regions may alter the cytoplasm composition, while directional flow
of this modified cytoplasm may affect the plasma membrane and chloroplast activities in cell regions residing downstream of
the illumination area. The impact of local illumination is predicted to be asymmetric because the cell regions located downstream
and upstream in the cytoplasmic flow with respect to illumination area would be exposed to flowing cytoplasm whose solute
composition was influenced by photosynthetic or dark metabolism. This hypothesis was checked by measuring H +-transporting activity of plasmalemma and chlorophyll fluorescence of chloroplasts in shaded regions of Chara corallina internodal cells near opposite borders of illuminated region (white light, beam width 2 mm). Both the apoplastic pH and chlorophyll
fluorescence, recorded in shade regions at equal distances from illuminated area, exhibited asymmetric light-on responses
depending on orientation of cytoplasmic streaming at the light–shade boundary. In the region where the cytoplasm flowed from
illuminated area to the measurement area, the alkaline zone (a zone with high plasma membrane conductance) was formed within
4-min illumination, whereas no alkaline zone was observed in the area where cytoplasm approached the boundary from darkened
regions. The results emphasize significance of cyclosis in lateral distribution of a functionally active intermediate capable
of affecting the membrane transport across the plasmalemma, the functional activity of chloroplasts, and pattern formation
in the plant cell. 相似文献
4.
The exchange properties of the activator CO 2 of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase were characterized both in vitro with the purified enzyme, and in situ within isolated chloroplasts. Carboxyarabinitol-1,5-bisphosphate, a proposed reaction intermediate analog for the carboxylase activity of the enzyme, was used to trap the activator CO 2 on the enzyme both in vitro and in situ. Modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in intact chloroplasts during a light/dark cycle was associated with a similar modulation in carboxyarabinitol-1,5-bisphosphate-trapped CO 2. The exchange kinetics of the activator CO 2 were monitored by activation of the enzyme to steady state in the presence of 12CO 2, followed by addition of 14CO 2 and determination of the amount of labeled CO 2 trapped on the enzyme by carboxyarabinitol-1,5-bisphosphate. Rate constants ( Kobs) for exchange with both the purified enzyme (0.45 min −1) and in illuminated chloroplasts (0.18 min −1) were comparable to the observed rate constants for enzyme activation under the two conditions. A similar exchange of the activator CO 2 was not observed in chloroplasts in the dark. Kinetic analysis of the exchange properties of the purified enzyme were consistent with an equilibrium between active and inactive forms of the enzyme during steady state activation. 相似文献
5.
Local illumination of the characean internode with a 30-s pulse of white light was found to induce the delayed transient increase of modulated chlorophyll fluorescence in shaded cell parts, provided the analyzed region is located downstream in the cytoplasmic flow at millimeter distances from the light spot. The fluorescence response to photostimulation of a remote cell region indicates that the metabolites produced by source chloroplasts in an illuminated region are carried downstream with the cytoplasmic flow, thus ensuring long-distance communications between anchored plastids in giant internodal cells. The properties of individual stages of metabolite signaling are not yet well known. We show here that the export of assimilates and/or reducing equivalents from the source chloroplasts into the flowing cytoplasm is largely insensitive to the direction of plasma-membrane H + flows, whereas the events in sink regions where these metabolites are delivered to the acceptor chloroplasts under dim light are controlled by H + fluxes across the plasma membrane. The fluorescence response to local illumination of remote cell regions was best pronounced under weak background light and was also observed in a modified form within 1–2 min after the transfer of cell to darkness. The fluorescence transients in darkened cells were suppressed by antimycin A, an inhibitor of electron transfer from ferredoxin to plastoquinone, whereas the fluorescence response under background light was insensitive to this inhibitor. We conclude that the accumulation of reduced metabolites in the stroma leads to the reduction of photosystem II primary quinone acceptor (Q A) via two separate (photochemical and non-photochemical) pathways. 相似文献
6.
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 CO 2 was optimum. Isolated chloroplasts were about 90% intact by microscopic examination, ferricyanide-dependent O 2 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 O 2 and fixed 21 micromoles of CO 2 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 CO 2 in air for 24 hours. On low CO 2 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. 相似文献
7.
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
8.
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
9.
《BBA》2020,1861(10):148257
Export of reducing power from chloroplasts to cytoplasm serves to balance the NADPH/ATP ratio that is optimal for CO 2 assimilation. Rapid cytoplasmic streaming in characean algae conveys the exported metabolites downstream towards the shaded plastids where envelope transporters may operate for the import of reducing power in accordance with the direction of concentration gradients. Import of reducing equivalents by chloroplasts in the analyzed area transiently enhances the pulse-modulated chlorophyll fluorescence F′ controlled by the redox state of photosystem II acceptor Q A. When the microfluidic pathway was transferred to darkness while the analyzed cell area remained in dim background light, the amplitude of cyclosis-mediated F′ changes dropped sharply and then recovered within 5–10 min. The suppression of long-distance signaling indicates temporal depletion of transmitted metabolites in the streaming cytoplasm. The return to overall background illumination induced an exceptionally large F′ response to the first local light pulse admitted to a remote cell region. This indicates the appearance of excess reductants in the streaming cytoplasm at a certain stage of photosynthetic induction. The results suggest highly dynamic exchange of metabolites between stationary chloroplasts lining the microfluidic pathway and the streaming cytoplasm upon light–dark and dark–light transitions. Evidence is obtained that slow stages of chlorophyll fluorescence induction in algae with rapid cytoplasmic streaming directly depend on cyclosis-mediated long-distance delivery of metabolites produced far beyond the analyzed cell area. 相似文献
10.
Magnesium was most inhibitory to photosynthetic reactions by intact chloroplasts when the magnesium was added in the dark before illumination. Two millimolar MgCl 2, added in the dark, inhibited CO 2-dependent O 2 evolution by Hordeum vulgare L. and Spinacia oleracea L. (C 3 plants) chloroplasts 70 to 100% and inhibited (pyruvate + oxaloacetate)-dependent O 2 evolution by Digitaria sanguinalis L. (C 4 plant) mesophyll chloroplasts from 80 to 100%. When Mg 2+ was added in the light, O 2 evolution was reduced only slightly. O 2 evolution in the presence of phosphoglycerate was less sensitive to Mg 2+ inhibition than was CO 2-dependent O 2 evolution. Magnesium prevented the light activation of several photosynthetic enzymes. Two millimolar Mg2+ blocked the light activation of NADP-malate dehydrogenase in D. sanguinalis mesophyll chloroplasts, and the light activation of phosphoribulokinase, NADP-linked glyceraldehyde-3-phosphate dehydrogenase, and fructose 1,6-diphosphatase in barley chloroplasts. The results suggest that Mg2+ inhibits chloroplast photosynthesis by preventing the light activation of certain enzymes. 相似文献
11.
Because glyoxylate inhibits CO 2 fixation by intact chloroplasts and purified ribulose bisphosphate carboxylase/oxygenase, glyoxylate might be expected to exert some regulatory effect on photosynthesis. However, ribulose bisphosphate carboxylase activity and activation in intact chloroplasts from Spinacia oleracea L. leaves were not substantially inhibited by 10 millimolar glyoxylate. In the light, the ribulose bisphosphate pool decreased to half when 10 millimolar glyoxylate was present, whereas this pool doubled in the control. When 10 millimolar glyoxylate or formate was present during photosynthesis, the fructose bisphosphate pool in the chloroplasts doubled. Thus, glyoxylate appeared to inhibit the regeneration of ribulose bisphosphate, but not its utilization. The fixation of CO2 by intact chloroplasts was inhibited by salts of several weak acids, and the inhibition was more severe at pH 6.0 than at pH 8.0. At pH 6.0, glyoxylate inhibited CO2 fixation by 50% at 50 micromolar, and glycolate caused 50% inhibition at 150 micromolar. This inhibition of CO2 fixation seems to be a general effect of salts of weak acids. Radioactive glyoxylate was reduced to glycolate by chloroplasts more rapidly in the light than in the dark. Glyoxylate reductase (NADP+) from intact chloroplast preparations had an apparent Km (glyoxylate) of 140 micromolar and a Vmax of 3 micromoles per minute per milligram chlorophyll. 相似文献
12.
Aerated and stirred suspensions of mechanically isolated Asparagus sprengeri Regel mesophyll cells were used to investigate the roles of respiration and photosynthesis in net H + efflux. Rates varied between 0.12 and 1.99 nanomoles H + per 10 6 cells per minute or 3 and 40 nanomoles H + per milligram chlorophyll per minute. The mean rate of H + efflux was 10% greater in the dark. 3-(3,4-Dichlorophenyl)-l,l-dimethylurea, an inhibitor of noncyclic photophosphorylation, did not inhibit H + efflux from illuminated cells. Bubbling with N 2 or addition of oligomycin, an inhibitor of mitochondrial ATP production, resulted in rapid and virtually complete inhibition of H + efflux in light or dark. In the absence of aeration, H + efflux came to a halt but resumed with aeration or illumination. When aeration was switched to CO 2-free air, rates of H + efflux were reduced 43% in the dark and 57% in the light. Oligomycin eliminated dark CO 2 fixation but not photosynthetic CO 2 fixation. It is suggested that H + efflux is dependent on respiration and dark CO 2 fixation, but independent of photosynthesis. 相似文献
13.
Cyanide and azide inhibit photosynthesis and catalase activity of isolated, intact spinach ( Spinacia oleracea) chloroplasts. When chloroplasts are illuminated in the presence of CN − or N 3−, accumulation of H 2O 2 is observed, parallel to inhibition of photosynthesis. Photosynthetic O 2 evolution is inhibited to the same extent, under saturating light, whether CO 2 or phosphoglycerate is present as electron acceptor. 相似文献
14.
The light-dependent accumulation of radioactively labeled inorganic carbon in isolated spinach ( Spinacia oleracea L.) chloroplasts was determined by silicone oil filtering centrifugation. Intact chloroplasts, dark-incubated 60 seconds at pH 7.6 and 23°C with 0.5 millimolar sodium bicarbonate, contained 0.5 to 1.0 millimolar internal inorganic carbon. The stromal pool of inorganic carbon increased 5- to 7-fold after 2 to 3 minutes of light. The saturated internal bicarbonate concentration of illuminated spinach chloroplasts was 10- to 20-fold greater than that of the external medium. This ratio decreased at lower temperatures and with increasing external bicarbonate. Over one-half the inorganic carbon found in intact spinach chloroplasts after 2 minutes of light was retained during a subsequent 3-minute dark incubation at 5°C. Calculations of light-induced stromal alkalization based on the uptake of radioactively labeled bicarbonate were 0.4 to 0.5 pH units less than measurements performed with [ 14C]dimethyloxazolidine-dione. About one-third of the binding sites on the enzyme ribulose 1,5-bisphosphate carboxylase were radiolabeled when the enzyme was activated in situ and 14CO 2 bound to the activator site was trapped in the presence of carboxypentitol bisphosphates. Deleting orthophosphate from the incubation medium eliminated inorganic carbon accumulation in the stroma. Thus, bicarbonate ion distribution across the chloroplast envelope was not strictly pH dependent as predicted by the Henderson-Hasselbach formula. This finding is potentially explained by the presence of bound CO 2 in the chloroplast. 相似文献
15.
Leaves of maize ( Zea mays L.) and sugar beet ( Beta vulgaris L.) were enclosed in an illuminated chamber in air for 30 min after which time 14CO 2 was released into the chamber. Two min after the 14CO 2 was released, the leaves were removed from the chamber, and small sections were cut from them. The sections were put in small wire baskets and frozen in isopentane cooled by liquid nitrogen. Approximately 1.5 min elapsed from the removal of the leaf from the illuminated chamber until the tissue was frozen. The tissue was freeze-dried, embedded in paraffin and the cellular location of the isotopic activity was determined by radiography of leaf cross sections. Isotopic activity in maize leaves was localized in bundle sheath parenchyma. In contrast, the label in sugar beet leaves was generally distributed in the mesophyll cells. The bundle sheath cells in maize contain specialized chloroplasts which appear to have a unique capacity to incorporate CO 2. Translocation from leaves of maize was 3-fold as rapid as from sugar beet leaves in the same environment. Low light intensity did not alter the distribution pattern of fixed CO 2. 相似文献
16.
9-Aminoacridine has been used to monitor the intrathylakoid pH of photo-synthetically competent intact chloroplasts. Values obtained from 9-aminoacridine accumulation in the chloroplasts must be corrected for light-dependent binding of 9-aminoacridine to the thylakoid membranes. During nitrite reduction by intact chloroplasts, the intrathylakoid proton concentration increased. It decreased somewhat during CO 2 reduction. However, low concentrations of uncoupling amines such as NH 3 or cyclohexylamine, which rapidly penetrated the chloroplast envelope and decreased the intrathylakoid proton concentration, failed to reduce, and actually stimulated, rates of CO 2-dependent oxygen evolution even under rate-limiting light. In contrast, low concentrations of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or nigericin, which inhibited CO 2 reduction, even appeared to increase the intrathylakoid proton concentration. As indicated by measurements of the 515 nm signal of the chloroplasts, the light-induced membrane potential was not much affected by low concentrations of the uncoupling amines, but was decreased by FCCP and by high concentrations of the amines. Even in the presence of high concentrations of NH 4Cl, ATP/ADP ratios of illuminated chloroplasts remained far above the ratios observed in the dark. In contrast, low concentrations of FCCP were sufficient to reduce ATP/ADP ratios to the dark value even under high intensity illumination. The observations are difficult to explain within the framework of the chemiosmotic hypothesis as presently discussed. 相似文献
17.
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 ( 1O 2) scavengers, histidine and sodium azide (NaN 3) significantly protected against the damage caused to photosystem II. The hydroxyl radical scavenger formate failed to protect the thylakoid membranes. The production of 1O 2 monitored as N,N-dimethyl p-nitrosoaniline bleaching increased as a function of light exposure time of treated chloroplasts and was abolished by the 1O 2 quencher, NaN 3. Membrane lipid peroxidation monitored as malondialdehyde production was also significantly reduced when chloroplasts were illuminated in the presence of NaN 3 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. 相似文献
18.
When isolated spinach chloroplasts are illuminated, photosynthesis and CO 2 fixation die off within 30 to 90 minutes. Even when air levels of CO 2 are used which maintain high and rate-saturating amounts of ribulose 1,5-bisphosphate inside the plastids, CO 2 fixation declines. The decline begins with a drop in activity of the ribulose 1,5-bishosphate carboxylase/oxygenase, specifically loss of the enzyme-activator CO 2-Mg 2+ form. Next, the light reactions cause gradual leakage of the carboxylase and other stromal proteins to the suspending medium. The chloroplast outer envelope appears to reseal and protect the thylakoids since there is little change in the ferricyanide-dependent Hill reaction. In the dark, under otherwise identical conditions, leakage of carboxylase does not occur. 相似文献
19.
Carbon transport across the plasma membrane, and carbon fixation were measured in perfused Chara internodal cells. These parameters were measured in external media of pH 5·5 and pH 8·5, where CO 2 and HCO 3- are, respectively, the predominant carbon species in both light and dark conditions. Cells perfused with medium containing ATP could utilize both CO 2 and HCO 3- from the external medium in the light. Photosynthetic carbon fixation activity was always higher at pH 5·5 than at pH 8·5. When cells were perfused either with medium containing hexokinase and 2-deoxyglucose to deplete ATP from the cytosol (HK medium) or with medium containing vanadate, a specific inhibitor of the plasma membrane H +-ATPase (V medium), photosynthetic carbon fixation was strongly inhibited at both pH 5·5 and 8·5. Perfusion of cells with medium containing pyruvate kinase and phosphoenolpyruvate (PEP) to maximally activate the H +-ATPase (PK medium), stimulated the photosynthetic carbon fixation activities. Oxygen evolution of isolated chloroplasts and the carbon fixation of cells supplied 14C intracellularly were not inhibited by perfusion media containing either hexokinase and 2-deoxyglucose or vanadate. The results indicate that Chara cells possess CO 2 and HCO 3- transport systems energized by ATP and sensitive to vanadate in the light. In the dark, intact cells also fix carbon. By contrast, in cells perfused with medium containing ATP, no carbon fixation was detected in 1 mol m -3 total dissolved inorganic carbon (TDIC) at pH 8·5. By increasing TDIC to 10 mol m -3, dark fixation became detectable, although it was still lower than that of intact cells at 1mol m -3 TDIC. Addition of PEP or PEP and PEP carboxylase to the perfusion media significantly increased the dark-carbon fixation. Perfusion with vanadate had no effect on the dark-carbon fixation. 相似文献
20.
Potential competition between CO 2 and NO 2− photoassimilation for photogenerated reductant ( e.g. reduced ferredoxin and NADPH) was examined employing isolates of mesophyll cells and intact chloroplasts derived from mature `source' spinach leaves. Variations in the magnitude of incident light energy were used to manipulate the supply of reductant in situ within chloroplasts. Leaf cell and plastid isolates were fed with saturating CO 2 and/or NO 2− to produce the highest demand for reductant by CO 2 and/or NO 2− assimilatory processes (enzymes). Even in the presence of CO 2 fixation, NO 2− reduction in intact leaf cell isolates as well as plastid isolates was maximal at light energies as low as 50 to 200 microeinsteins per second per square meter. Simultaneously, 500 to 800 microeinsteins per second per square meter were required to support maximal CO 2 assimilation. Regardless of the magnitude of the incident light energy, CO 2 assimilation did not repress NO 2− reduction, nor were these two processes mutually repressed. These observations have been interpreted to mean that reduced ferredoxin levels in situ in the plastids of mature source leaf mesophyll cells were adequate to supply the concurrent maximal demands exerted by enzymes associated with CO 2 as well as with inorganic nitrogen photoassimilation. 相似文献
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