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1.
Active CO(2) Transport by the Green Alga Chlamydomonas reinhardtii   总被引:6,自引:6,他引:0       下载免费PDF全文
Mass spectrometric measurements of dissolved free 13CO2 were used to monitor CO2 uptake by air grown (low CO2) cells and protoplasts from the green alga Chlamydomonas reinhardtii. In the presence of 50 micromolar dissolved inorganic carbon and light, protoplasts which had been washed free of external carbonic anhydrase reduced the 13CO2 concentration in the medium to close to zero. Similar results were obtained with low CO2 cells treated with 50 micromolar acetazolamide. Addition of carbonic anhydrase to protoplasts after the period of rapid CO2 uptake revealed that the removal of CO2 from the medium in the light was due to selective and active CO2 transport rather than uptake of total dissolved inorganic carbon. In the light, low CO2 cells and protoplasts incubated with carbonic anhydrase took up CO2 at an apparently low rate which reflected the uptake of total dissolved inorganic carbon. No net CO2 uptake occurred in the dark. Measurement of chlorophyll a fluorescence yield with low CO2 cells and washed protoplasts showed that variable fluorescence was mainly influenced by energy quenching which was reciprocally related to photosynthetic activity with its highest value at the CO2 compensation point. During the linear uptake of CO2, low CO2 cells and protoplasts incubated with carbonic anhydrase showed similar rates of net O2 evolution (102 and 108 micromoles per milligram of chlorophyll per hour, respectively). The rate of net O2 evolution (83 micromoles per milligram of chlorophyll per hour) with washed protoplasts was 20 to 30% lower during the period of rapid CO2 uptake and decreased to a still lower value of 46 micromoles per milligram of chlorophyll per hour when most of the free CO2 had been removed from the medium. The addition of carbonic anhydrase at this point resulted in more than a doubling of the rate of O2 evolution. These results show low CO2 cells of Chlamydomonas are able to transport both CO2 and HCO3 but CO2 is preferentially removed from the medium. The external carbonic anhydrase is important in the supply to the cells of free CO2 from the dehydration of HCO3.  相似文献   

2.
High rates of both cyclic and noncyclic photophosphorylation were measured in chloroplast lamellae isolated from purified guard cell protoplasts from Vicia faba L. Typical rates of light-dependent incorporation of 32P into ATP were 100 and 190 micromoles ATP per milligram chlorophyll per hour for noncyclic (water to ferricyanide) and cyclic (phenazine methosulfate) photophosphorylation, respectively. These rates were 50 to 80% of those observed with mesophyll chloroplasts. Noncyclic photophosphorylation in guard cell chloroplasts was completely inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea supporting the notion that photophosphorylation is coupled to linear electron flow from photosystem II to photosystem I. Several lines of evidence indicated that contamination by mesophyll chloroplasts cannot account for the observed photophosphorylation rates.

A comparison of the photon fluence dependence of noncyclic photophosphorylation in mesophyll and guard cell chloroplasts showed significant differences between the two preparations, with half saturation at 0.04 and 0.08 millimole per square meter per second, respectively.

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3.
Aerobic and anaerobic respiration in the intact spinach chloroplast   总被引:3,自引:3,他引:0       下载免费PDF全文
Aerobic and anaerobic chloroplastic respiration was monitored by measuring 14CO2 evolution from [14C]glucose in the darkened spinach (Spinacia oleracea) chloroplast and by estimating the conversion of fructose 1,6-bisphosphate to glycerate 3-phosphate in the darkened spinach chloroplast in air with O2 or in N2 with nitrite or oxaloacetate as electron acceptors. The pathway of 14CO2 evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide and glycolate 2-phosphate under air or N2 were those expected from the oxidative pentose phosphate cycle and glycolysis. Of the electron acceptors, O2 was the best (2.4 nanomoles CO2 per milligram chlorophyll per hour), followed by nitrite and oxaloacetate. With respect to glycerate 3-phosphate formation from fructose 1,6-bisphosphate, methylene blue increased the aerobic rate from 3.7 to 5.4 micromoles per milligram chlorophyll per hour. A rate of 4.8 micromoles per milligram chlorophyll per hour was observed under N2 with nitrite and oxaloacetate.  相似文献   

4.
Robinson JM  Gibbs M 《Plant physiology》1982,70(5):1249-1254
Light-dependent O2 reduction concomitant with O2 evolution, ATP formation, and NADP reduction were determined in isolated spinach (Spinacia oleracea L. var. America) chloroplast lamellae fortified with NADP and ferredoxin. These reactions were investigated in the presence or absence of catalase, providing a tool to estimate the reduction of O2 to H2O2 (Mehler reaction) concomitant with NADP reduction. In the presence of 250 micromolar O2, O2 photoreduction, simultaneous with NADP photoreduction, was dependent upon light intensity, ferredoxin, Mn2+, NADP, and the extent of coupling of phosphorylation to electron flow.

In the presence of an uncoupling concentration of NH4+, saturating light intensity (>500 watts/square meter), saturating ferredoxin (10 micromolarity) rate-limiting to saturating NADP (0.2-0.9 millimolarity), and Mn2+ (50-1000 micromolarity), the maxium rates of O2 reduction were 13-25 micromoles/milligram chlorophyll per hour, while concomitant rates of O2 evolution and NADP reduction were 69 to 96 and 134 to 192 micromoles/milligram chlorophyll per hour, respectively. Catalase did not affect the rate of NADPH or ATP formation but decreased the NADPH:O2 ratios from 2.3-2.8 to 1.9-2.1 in the presence of rate-limiting as well as saturating concentrations of NADP.

Photosynthetic electron flow at a rate of 31 micromoles O2 evolved/milligram chlorophyll per hour was coupled to the synthesis of 91 micromoles ATP/milligram chlorophyll per hour, while the concomitant rate of O2 reduction was 0.6 micromoles/milligram chlorophyll per hour and was calculated to be associated with an apparent ATP formation of only 2 micromoles/milligram chlorophyll per hour. Thus, electron flow from H2O to O2 did not result in ATP formation significantly above that produced during NADP reduction.

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5.
Mesophyll cells and bundle sheath strands were isolated rapidly from leaves of the C4 species Digitaria pentzii Stent. (slenderstem digitgrass) by a chopping and differential filtration technique. Rates of CO2 fixation in the light by mesophyll and bundle sheath cells without added exogenous substrates were 6.3 and 54.2 micromoles of CO2 per milligram of chlorophyll per hour, respectively. The addition of pyruvate or phosphoenolpyruvate to the mesophyll cells increased the rates to 15.2 and 824.6 micromoles of CO2 per milligram of chlorophyll per hour, respectively. The addition of ribose 5-phosphate increased the rate for bundle sheath cells to 106.8 micromoles of CO2 per milligram of chlorophyll per hour. These rates are comparable to those reported for cells isolated by other methods. The Km(HCO3) for mesophyll cells was 0.9 mm; for bundle sheath cells it was 1.3 mm at low, and 40 mm at higher HCO3 concentrations. After 2 hours of photosynthesis by mesophyll cells in 14CO2 and phosphoenolpyruvate, 88% of the incorporated 14C was found in organic acids and 0.8% in carbohydrates; for bundle sheath cells incubated in ribose 5-phosphate and ATP, more than 58% of incorporated 14C was found in carbohydrates, mainly starch, and 32% in organic acids. These findings, together with the stimulation of CO2 fixation by phosphoenolpyruvate for mesophyll cells and by ribose 5-phosphate plus ATP for bundle sheath cells, and the location of phosphoenolpyruvate and ribulose bisphosphate carboxylases in mesophyll and bundle sheath cells, respectively, are in accord with the scheme of C4 photosynthesis which places the Calvin cycle in the bundle sheath and C4 acid formation in mesophyll cells.  相似文献   

6.
Isolation of mesophyll protoplasts from mature leaves of soybeans   总被引:3,自引:2,他引:1       下载免费PDF全文
Lin W 《Plant physiology》1983,73(4):1067-1069
A procedure based on a combined cellulase-Pectolyase Y-23 enzyme digestion and metrizamide-sorbitol gradient purification protocol was developed for isolating mesophyll protoplasts from mature leaves of soybean (Glycine max L. Merr.). Based on chlorophyll content, this procedure results in a 10 to 15% protoplast yield from fully expanded mature leaves and a 20 to 30% yield from young (expanding) leaves within 3 hours. Isolated protoplasts displayed high rates of HCO3-dependent photosynthesis; greater than 75 micromoles O2 evolved per milligram chlorophyll per hour at 25°C. This photosynthetic rate is comparable to that of mesophyll cells isolated mechanically from the same leaves.  相似文献   

7.
Previously, C Baysdorfer and JM Robinson (1985 Plant Physiol 77: 318-320) demonstrated that, in a reconstituted spinach chloroplast system, NADP photoreduction functioning at most maximal rate and reductant demand, was the successful competitor with NO2 photoreduction for reduced ferredoxin. This resulted in a repression of NO2 reduction until all NADP available had been almost totally reduced. Further experiments, employing isolated, intact spinach leaf plastids and soybean leaf mesophyll cells, were conducted to examine competition for reductant between CO2 and NO2 photoassimilation, in situ. In isolated, intact plastid preparations, regardless of whether the demand for reductant by CO2 photoassimilation was high (5 millimolar `CO2') with rates of CO2 fixation in the range 40 to 90 micromoles CO2 fixed per hour per milligram chlorophyll, low (0.5 millimolar `CO2') with rates in the range 5 to 8 micromoles CO2 per hour per milligram chlorophyll, or zero (no `CO2'), NO2 photoreduction displayed equal rates in the range of 8 to 22 micromoles per hour per milligram chlorophyll. In the absence of `CO2', but in the presence of saturating white light, 3-phosphoglycerate photoreduction at rates of 82 to 127 micromoles per hour per milligram chlorophyll did not repress, and occasionally stimulated concomitant rates of NO2 reduction which ranged from 23.4 to 38.5. Conversely, in plastid preparations, NO2 at levels of 50 to 100 micromolar, stimulated plastid CO2 fixation when `CO2' was saturating with respect to carboxylation. Further, levels of NO2 in the range 250 to 2500 micromolar, stimulated soybean leaf mesophyll cell net CO2 fixation as much as 1.5-fold if `CO2' was saturating with respect to CO2 fixation. It appeared likely that, in high light in vivo, CO2 and NO2 photoassimilatory processes are not forced to intercompete for reduced ferredoxin in the intact chloroplast.  相似文献   

8.
Rotatore C  Colman B 《Plant physiology》1990,93(4):1597-1600
Chloroplasts, isolated from protoplasts of the green alga, Chlorella ellipsoidea, were estimated to be 99% intact by the ferricyanide-reduction assay, and gave CO2 and PGA-dependent rates of O2 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 CO2 supply from the uncatalyzed dehydration of HCO3 indicating a direct uptake of bicarbonate by the intact chloroplasts. Mass spectrometric measurements of CO2 depletion from the medium on the illumination of chloroplasts indicate the lack of an active CO2 transport across the chloroplast envelope.  相似文献   

9.
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: CO2 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   

10.
Intact mesophyll and bundle sheath chloroplasts wee isolated from the NADP-malic enzyme type C4 plants maize, sorghum (monocots), and Flaveria trinervia (dicot) using enzymic digestion and mechanical isolation techniques. Bundle sheath chloroplasts of this C4 subgroup tend to be agranal and were previously reported to be deficient in photosystem II activity. However, following injection of intact bundle sheath chloroplasts into hypotonic medium, thylakoids had high Hill reaction activity, similar to that of mesophyll chloroplasts with the Hill oxidants dichlorophenolindophenol, p-benzoquinone, and ferricyanide (approximately 200 to 300 micromoles O2 evolved per mg chlorophyll per hour). In comparison to that of mesophyll chloroplasts, the Hill reaction activity of bundle sheath chloroplasts of maize and sorghum was labile and lost activity during assay. Bundle sheath chloroplasts of maize also exhibited some capacity for 3-phosphoglycerate dependent O2 evolution (29 to 58 micromoles O2 evolved per milligram chlorophyll per hour). Both the mesophyll and bundle sheath chloroplasts were equally effective in light dependent scavenging of hydrogen peroxide. The results suggest that both chloroplast types have noncyclic electron transport and the enzymology to reduce hydrogen peroxide to water. The activities of ascorbate peroxidase from these chloroplast types was consistent with their capacity to scavenge hydrogen peroxide.  相似文献   

11.
Activities of ribulose-1,5-bisphosphate carboxylase and rates of photosynthetic O2 evolution were measured in guard-cell and mesophyll protoplasts from Vicia faba. The ribulose-1,5-bisphosphate carboxylase activity of guard-cell protoplasts was 30% of that of mesophyll protoplasts; however, the O2 evolution rate was 3 times higher in guard-cell protoplasts than in mesophyll protoplasts on a chlorophyll basis. When the dark-adapted, guard-cell protoplasts were illuminated by red light, O2 was evolved with an induction period, which became shorter when the protoplasts were reilluminated. High activity of irreversible NADP-glyceraldehyde-3-phosphate dehyrogenase was found in guard-cell protoplasts. Several lines of evidence revealed that there was virtually no contamination by mesophyll cells in guard-cell preparations. These results indicate that guard cells fix CO2 photosynthetically and imply that the cells utilize a considerable proportion of reducing equivalents from water for reactions other than CO2 fixation.  相似文献   

12.
Kruse T  Tallman G  Zeiger E 《Plant physiology》1989,91(4):1382-1386
A method for isolating guard cell protoplasts (GCP) from mechanically prepared epidermis of Vicia faba is described. Epidermis was prepared by homogenizing leaves in a Waring blender in a solution of 10% Ficoll, 5 millimolar CaCl2, and 0.1% polyvinylpyrrolidone 40 (PVP). Attached mesophyll and epidermal cells were removed by shaking epidermis in a solution of Cellulysin, mannitol, CaCl2, PVP, and pepstatin A. Cleaned epidermis was transferred to a solution of mannitol, CaCl2, PVP, pepstatin A, cellulase “Onozuka” RS, and pectolyase Y-23 for the isolation of GCP. Preparations made by this method included both adaxial and abaxial GCP and contained ≤0.017% mesophyll protoplasts, ≤0.6% mesophyll fragments, and no epidermal cell contaminants. Yields averaged 9 × 104 protoplasts/leaflet and 98 to 100% of the GCP excluded trypan blue, concentrated neutral red, and hydrolyzed fluorescein diacetate. Isolated GCP increased in diameter by 2.2 micrometers after incubation in darkness in 10 micromolar fusicoccin, 0.4 molar mannitol, 5 millimolar KCl, and 1 millimolar CaCl2. Illumination of GCP with 800 micromoles per square meter per second of red light resulted in alkalinization of their suspension medium. When 10 micromolar per square meter per second of blue light was superimposed onto the red light background, the medium acidified. Measurements of chlorophyll a fast fluorescence transients from isolated GCP indicated that GCP were capable of electron transport, and slow transients contained the “M” peak usually associated with a functional photosynthetic carbon reduction pathway.  相似文献   

13.
Young expanding spinach leaves exposed to 14CO2 under physiological conditions for up to 20 minutes assimilated CO2 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 CO2in vivo at a mean rate of 2.1 micromoles per milligram chlorophyll per hour. Intact spinach chloroplasts isolated from these leaves incorporated H14CO3 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   

14.
Robinson SP 《Plant physiology》1982,70(4):1032-1038
Uptake of d, l-glycerate into the chloroplast stroma has been studied using the technique of silicone oil filtering centrifugation. Glycerate uptake was 3 to 5 times higher in the light than in darkness, the stimulation by light being abolished by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. The pH optimum for uptake was 7.0 at 2°C and 8.5 at 20°C, but at all pH values the rate of uptake was higher at 20°C than at 2°C. Uptake was concentration dependent, saturating above 8 millimolar glycerate. At 2°C, the Km was 0.3 millimolar and the Vmax was 13 micromoles per milligram of chlorophyll per hour. At 20°C initial rates of glycerate uptake were higher than 40 micromoles per milligram of chlorophyll per hour.  相似文献   

15.
Intact chloroplasts were isolated from protoplasts of the Crassulacean acid metabolism plant Sedum praealtum D.C. Typical rates of CO2 fixation or CO2-dependent O2 evolution ranged from 20 to 30 micromoles per milligram chlorophyll per hour and could be stimulated 30 to 50% by several Calvin cycle intermediates. The pH optimum for CO2 fixation was 7.0 to 7.6 with considerable activity as low as pH 6.4. Low concentrations of orthophosphate (Pi) (optimum 0.4 millimolar) stimulated photosynthesis while high concentrations (5 millimolar) caused some inhibition. Both CO2 fixation and CO2-dependent O2 evolution exhibited a relatively long lag phase (4 to 6 minutes) which remained constant between 0.4 to 5 millimolar Pi. The lag phase could be decreased by addition of dihydroxyacetone-phosphate or ribose 5-phosphate. Further results are presented which suggest these chloroplasts have a functional phosphate translocator.  相似文献   

16.
Cohen WS 《Plant physiology》1989,91(3):1107-1111
The membrane-bound coupling factor of maize mesophyll thylakoids is a latent ATPase. Mg2+-ATPase activity can be induced in the light with either dithiothreitol or low concentrations of trypsin. Maize thylakoids that are activated with light plus trypsin exhibit considerably higher levels of activity in Na2SO3-dependent Mg2+-ATPase assays compared to thylakoids that are light and dithiothreitol activated (1400 micromoles per milligram of chlorophyll per hour versus 200 micromoles per milligram of chlorophyll per hour). Treatment with light and dithiothreitol or light plus trypsin were also required to demonstrate high levels of octyl glucoside-dependent Mg2+-ATPase activity in maize mesophyll thylakoids. Only small differences in octyl glucoside-dependent Mg2+-ATPase activity were observed in preparations that were activated in the light with either trypsin or dithiothreitol. Mg2+-ATPase activity can also be induced in maize mesophyll chloroplasts by illuminating intact preparations under appropriate conditions. Little or no ATPase activity was observed in the absence of illumination or in the presence of light plus methyl viologen. The active state decayed in the dark with a t½ of 6 to 7 minutes at room temperature. Based on the effect of the thiol oxidant, o-iodosobenzoate, and the uncoupler, nigericin, on the kinetics of deactivation of ATPase activity in intact maize chloroplasts, it appears that the activation process requires a transmembrane proton gradient and reduction of a key disulfide bridge in the gamma of chloroplast coupling factor one.  相似文献   

17.
Isolated spinach (Spinacia oleracea L. var. Bloomsdale) leaf protoplasts reduced nitrate at rates of 9 micromoles per milligram chlorophyll per hour in light with a 3- to 4-fold stimulation in the presence of HCO3. A similar stimulation of nitrate reduction in the absence of CO2 fixation was obtained by the addition of malate, oxaloacetate (OAA), phospho-3-glyceric acid (PGA), or dihydroxyacetone phosphate (DHAP). Stimulation by malate and DHAP was light-independent, while the PGA and OAA effect was light-dependent. Nitrate reduction was found to be coupled to the cytoplasmic oxidation of DHAP or malate. The PGA/DHAP and OAA/malate shuttle across the chloroplast envelope has been demonstrated to support CO2 fixation and/or nitrate reduction. The leaf protoplasts readily assimilated nitrate into amino-N in a stoichiometric relationship.  相似文献   

18.
A procedure is described for isolating photosynthetically active rhodoplasts (“red algal chloroplasts”) from the marine alga Griffithsia monilis. The rhodoplasts exhibited rates of CO2 fixation and CO2-dependent O2 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 O2 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   

19.
Methionine sulfoximine induced release of ammonia from illuminated cells of Ankistrodesmus braunii (Naegeli) Brunnth, in normal air, but less in air enriched to 3% CO2. In normal air, methionine sulfoximine also induced glycolate release. Addition of either glutamate, glycine, or serine suppressed glycolate release, whereas glutamate and glycine at the same time stimulated ammonia release. The results indicate that inhibition of glutamine synthetase and thereby inhibition of photorespiratory nitrogen cycling restricts the sink capacity for glycolate in the photorespiratory carbon cycle. An external supply of glutamate, glycine, or serine seems to stimulate glyoxylate transamination and thus partly restores the sink capacity. Calculations of total glycolate formation rates in air from glycolate and ammonia release rates in the presence of methionine sulfoximine and glutamate revealed values of approximately 20 micromoles glycolate per milligram chlorophyll per hour on the average. Similar calculations led to an estimated rate of photorespiratory ammonia release in air, in the absence of methionine sulfoximine, of about 10 micromoles per milligram chlorophyll per hour on the average, a value comparable to the primary nitrogen assimilation rate of 8 micromoles per milligram chlorophyll per hour.  相似文献   

20.
Isolation of Intact Chloroplasts from Dunaliella tertiolecta   总被引:10,自引:7,他引:3       下载免费PDF全文
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.  相似文献   

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