Characterization of an Electron Transport Pathway Associated with Glucose and Fructose Respiration in the Intact Chloroplasts of Chlamydomonas reinhardtii and Spinach

The role of an electron transport pathway associated with aerobic carbohydrate degradation in isolated, intact chloroplasts was evaluated. This was accomplished by monitoring the evolution of ¹⁴CO₂ from darkened spinach (Spinacia oleracea) and Chlamydomonas reinhardtii chloroplasts externally supplied with [¹⁴C]fructose and [¹⁴C]glucose, respectively, in the presence of nitrite, oxaloacetate, and conventional electron transport inhibitors. Addition of nitrite or oxaloacetate increased the release of ¹⁴CO₂, but it was shown that O₂ continued to function as a terminal electron acceptor. ¹⁴CO₂ evolution was inhibited up to 30 and 15% in Chlamydomonas and spinach, respectively, by 50 μm rotenone and by amytal, but at 500- to 1000-fold higher concentrations, indicating the involvement of a reduced nicotinamide adenine dinucleotide phosphate-plastoquinone oxidoreductase. ¹⁴CO₂ release from the spinach chloroplast was inhibited 80% by 25 μm 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. ¹⁴CO₂ release was sensitive to propylgallate, exhibiting approximately 50% inhibition in Chlamydomonas and in spinach chloroplasts of 100 and 250 μm concentrations, respectively. These concentrations were 20- to 50-fold lower than the concentrations of salicylhydroxamic acid (SHAM) required to produce an equivalent sensitivity. Antimycin A (100 μm) inhibited approximately 80 to 90% of ¹⁴CO₂ release from both types of chloroplast. At 75 μm, sodium azide inhibited ¹⁴CO₂ evolution about 50% in Chlamydomonas and 30% in spinach. Sodium azide (100 mm) combined with antimycin A (100 μm) inhibited ¹⁴CO₂ evolution more than 90%. ¹⁴CO₂ release was unaffected by uncouplers. These results are interpreted as evidence for a respiratory electron transport pathway functioning in the darkened, isolated chloroplast. Chloroplast respiration defined as ¹⁴CO₂ release from externally supplied [1-¹⁴C]glucose can account for at least 10% of the total respiratory capacity (endogenous release of CO₂) of the Chlamydomonas reinhardtii cell.     

Glucose Respiration in the Intact Chloroplast of Chlamydomonas reinhardtii

Chloroplastic respiration was monitored by measuring ¹⁴CO₂ from ¹⁴C glucose in the darkened Chlamydomonas reinhardtii F-60 chloroplast. The patterns of ¹⁴CO₂ evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide, glycolate-2-phosphate, and phosphoenolpyruvate were those expected from the oxidative pentose phosphate cycle and glycolysis. The K_m for glucose was 56 micromolar and for MgATP was 200 micromolar. Release of ¹⁴CO₂ was inhibited by phloretin and inorganic phosphate. Comparing the inhibition of CO₂ evolution generated by pH 7.5 with respect to pH 8.2 (optimum) in chloroplasts given C-1, C-2, and C-6 labeled glucose indicated that a suboptimum pH affects the recycling of the pentose phosphate intermediates to a greater extent than CO₂ evolution from C-1 of glucose. Respiratory inhibition by pH 7.5 in the darkened chloroplast was alleviated by NH₄Cl and KCl (stromal alkalating agents), iodoacetamide (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), or phosphoenolpyruvate (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiration in the darkened Chlamydomonas chloroplast is the fructose-1,6-bisphosphatase/phosphofructokinase junction. The respiratory pathways described here can account for the total oxidation of a hexose to CO₂ and for interactions between carbohydrate metabolism and the oxyhydrogen reaction in algal cells adapted to a hydrogen metabolism.     

Respiration of Sugars in Spinach (Spinacia oleracea), Maize (Zea mays), and Chlamydomonas reinhardtii F-60 Chloroplasts with Emphasis on the Hexose Kinases

The role of hexokinase in carbohydrate degradation in isolated, intact chloroplasts was evaluated. This was accomplished by monitoring the evolution of 14CO2 from darkened spinach (Spinacia oleracea), maize (Zea mays) mesophyll, and Chlamydomonas reinhardtii chloroplasts externally supplied with 14C-labeled fructose, glucose, mannose, galactose, maltose, and ribose. Glucose and ribose were the preferred substrates with the Chlamydomonas and maize chloroplasts, respectively. The rate of CO2 release from fructose was about twice that from glucose in the spinach chloroplast. Externally supplied ATP stimulated the rate of CO2 release. The pH optimum for CO2 release was 7.5 with ribose and fructose and 8.5 with glucose as substrates. Probing the outer membrane polypeptides of the intact spinach chloroplast with two proteases, trypsin and thermolysin, decreased 14CO2 release from glucose about 50% but had little effect when fructose was the substrate. Tryptic digestion decreased CO2 release from glucose in the Chlamydomonas chloroplast about 70%. 14CO2 evolution from [1-14C]-glucose-6-phosphate in both chloroplasts was unaffected by treatment with trypsin. Enzymic analysis of the supernatant (stroma) of the lysed spinach chloroplast indicated a hexokinase active primarily with fructose but with some affinity for glucose. The pellet (membranal fraction) contained a hexokinase utilizing both glucose and fructose but with considerably less total activity than the stromal enzyme. Treatment with trypsin and thermolysin eliminated more than 50% of the glucokinase activity but had little effect on fructokinase activity in the spinach chloroplast. Tryptic digestion of the Chlamydomonas chloroplast resulted in a loss of about 90% of glucokinase activity.     

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.

     

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.     

Evidence for Inorganic Carbon Transport by Intact Chloroplasts of Chlamydomonas reinhardtii

Isolated intact chloroplasts from wall-less mutants of Chlamydomonas reinhardtii accumulate inorganic carbon (C_i) from the medium provided the cells had been adapted to low CO₂ photoautotrophic growth conditions. Chloroplasts from cultures grown on high (5%) CO₂ or photoheterotrophically with acetate did not accumulate inorganic carbon. Chloroplast C_i accumulation from low CO₂ grown cells was light dependent and was inhibited by uncouplers and inhibitors of electron transport. In a model for C_i accumulation by Chlamydomonas, it is proposed that CO₂ diffuses into the cell and C_i accumulation occurs in the chloroplast.     

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.     

低渗膨胀对菠菜完整叶绿体光合作用的影响

菠菜离体完整叶绿体需要合适的介质渗透压（约０．９ＭＰａ）以保持其较高的光合作用速率。当渗透压因降低介质中山梨醇浓度（从０．３３ｍｏｌ／Ｌ至０．１７ｍｏｌ／Ｌ）而降低时,叶绿体的完整率保持不变。低于临界渗透压（约０．５ＭＰａ）,叶绿体被膜就发生破裂．并丧失ＣＯ２同化能力。在轻度低渗条件下,虽然叶绿体被膜未破,但依赖ＣＯ２的放氧速率已受抑制。渗透压在０．９ＭＰａ与０．５ＭＰａ之间,叶绿体依赖ＰＧＡ的放氧抑制,可由加入山梨醇至正常浓度（０．３３ｍｏｌ／Ｌ）而解除。膨涨叶绿体的ＡＴＰ合成水平与正常叶绿体相同,而ＮＡＤＰＨ形成速率则明显降低。利用能透过被膜的不同电子受体ＮＣ２、ＰＧＡ和ＯＡＡ发现,在膨胀叶绿体中,ＮＯ２的还原不受形响,而ＰＧＡ及ＯＡＡ的还原明显被抑制。我们推测,低渗膨胀叶绿体中光合作用的抑制,至少有一个原因是Ｆｄ－ＮＡＤＰ氧化还原酶作用的受阻。     

Some Enzymes and Properties of the Reductive Carboxylic Acid Cycle Are Present in the Green Alga Chlamydomonas reinhardtii F-60

The reductive carboxylic acid cycle, the autotrophic pathway of CO₂ assimilation in prokaryotes (photosynthetic and nonphotosynthetic autotrophic bacteria), was investigated in Chlamydomonas reinhardtii F-60, an algal mutant lacking a complete photosynthetic carbon reduction pathway (C₃) due to a deficiency in phosphoribulokinase. Evidence was obtained consistent with the presence of the reductive carboxylic acid cycle in F-60. This conclusion is based on the fact that: (a) acetate approximately doubled CO₂ fixation in whole cells (4 micromoles per milligram chlorophyll per hour) and in chloroplasts (32 nanomoles per milligram chlorophyll per hour); and (b) pyruvate synthase, α-ketoglutarate synthase, and ATP-citrate lyase, three indicators of the cycle, were found in cell-free extracts.     

H(2) and CO(2) Evolution by Anaerobically Adapted Chlamydomonas reinhardtii F-60

Using manometric and enzymic techniques, H₂ and CO₂ evolution in darkness and light has been studied in the green alga Chlamydomonas reinhardtii F-60. F-60 is a mutant strain characterized by an incomplete photosynthetic carbon reduction cycle but an intact electron transport chain.     

草酰乙酸和苹果酸对菠菜叶片和完整叶绿体光合作用的影响

观测了OAA和MA对菠菜叶片和完整叶绿体光合作用的影响.结果显示,当叶片切块在20μmol/L的OAA存在时,其叶片的光合放氧速率增加了89%,经OAA处理的离体完整叶绿体的光合放氧速率增加了72%;当反应体系中存在有较高浓度的NaHCO3时,OAA的作用不明显.叶片经20 μmol/L的MA处理后,叶片光合放氧速率比对照高127%.用CO2分析仪观测了处理后叶片的净光合速率(Pn),结果显示,OAA和MA处理后的叶片Pn值分别是对照的117%和111%.对在C3植物中建立C4微循环系统来提高光合作用效率的可能性进行了讨论.     

Salt-Induced Dissociation of Carbonic Anhydrase from Intact Cells of Chlamydomonas reinhardtii

The extracellular carbonic anhydrase of Chlamydomonas reinhardtii is dissociated from either intact or lysed cells by treatment with a 20 millimolar potassium phosphate buffer containing 0.4 molar KCI at pH 7.4. Electrophoretic analysis of proteins dissociated by the high salt treatment reveals that carbonic anhydrase comprises over 70% of the total released. These results suggest that the extracellular carbonic anhydrase in C. reinhardtii is bound to either the cell wall or plasma membrane through ionic interactions.     

Interactions between Photosynthesis and Respiration in the Green Alga Chlamydomonas reinhardtii (Characterization of Light-Enhanced Dark Respiration)

The rate of respiratory O2 consumption by Chlamydomonas reinhardtii cell suspensions was greater after a period of photosynthesis than in the preceding dark period. This "light-enhanced dark respiration" (LEDR) was a function of both the duration of illumination and the photon fluence rate. Mass spectrometric measurements of gas exchange indicated that the rate of gross respiratory O2 consumption increased during photosynthesis, whereas gross respiratory CO2 production decreased in a photon fluence rate-dependent manner. The rate of postillumination O2 consumption provided a good measure of the O2 consumption rate in the light. LEDR was substantially decreased by the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or glycolaldehyde, suggesting that LEDR was photosynthesis-dependent. The onset of photosynthesis resulted in an increase in the cellular levels of phosphoglycerate, malate, and phosphoenolpyruvate, and a decrease in whole-cell ATP and citrate levels; all of these changes were rapidly reversed upon darkening. These results are consistent with a decrease in the rate of respiratory carbon flow during photosynthesis, whereas the increase in respiratory O2 consumption during photosynthesis may be mediated by the export of photogenerated reductant from the chloroplast. We suggest that photosynthesis interacts with respiration at more than one level, simultaneously decreasing the rate of respiratory carbon flow while increasing the rate of respiratory O2 consumption.     

Effect of Disalicylidenepropanediamine on the Light-dependent Reduction of Carbon Dioxide and Glycerate 3-Phosphate in Intact Spinach Chloroplasts

Disalicylidenepropanediamine (DSPD) at 0.1 to 1 mm levels inhibited light-dependent (14)CO(2) assimilation in intact spinach chloroplasts about 50 to 80%, and this inhibition was accompanied by an increased ratio of (14)C-glycerate 3-phosphate to (14)C-glyceraldehyde 3-phosphate. Enzymatic analysis established that DSPD also inhibited the light-dependent reduction of glycerate 3-phosphate in intact spinach chloroplasts. DSPD at 0.5 mm did not inhibit ribose 5-phosphate isomerase, ribulose 5-phosphate kinase, glycerate 3-phosphate kinase, NADP(+)-linked glyceraldehyde 3-phosphate dehydrogenase or ribulose 1,5-diphosphate carboxylase. The inhibition of chloroplast (14)CO(2) assimilation by DSPD appeared to be related to the inhibition of the photosynthetic electron transport chain. These observations are consistent with experimental results which demonstrated that DSPD inhibited directly the chloroplast lamellar membrane-mediated, light-dependent reduction of ferredoxin (Trebst, A. and M. Burba, 1967, Z. Pflanzenphysiol. 57: 419-433 and Ben-Amotz, A. and M. Avron, 1972, Plant Physiol. 49: 244-248).     

Chloroplasts of the green alga Chlamydomonas reinhardtii possess at least four distinct stromal processing proteases

The majority of the proteins in the chloroplast are encoded in the nucleus and synthesised in the cytoplasm as precursors with N-terminal extensions. These targeting sequences guide the precursor proteins into the chloroplast where they are immediately cleaved off by a stromal processing protease (SPP). It is commonly assumed that in higher plant chloroplasts one general SPP processes almost all imported precursor proteins. In the green alga Chlamydomonas, however, there exist several different SPPs which process the various Chlamydomonas precursor proteins. The seven precursor proteins investigated here, which were all correctly imported into isolated chloroplasts, could be divided into two groups: Four precursor proteins were cleaved correctly when processed in vitro with an extract of stromal proteins. Four different SPPs were found in Chlamydomonas chloroplasts to be responsible for the processing of this class of precursors and these four activities were separated chromatographically, characterised and further distinguished by their sensitivity to different inhibitors. The three precursors of the second group were degraded completely by unidentified enzyme(s) present in the stromal extract. Degradation of these precursors was dependent on their conformational integrity as well as on the redox state in the stroma. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cytochrome and Alternative Pathway Respiration during Transient Ammonium Assimilation by N-Limited Chlamydomonas reinhardtii

Mass spectrometric analysis of gas exchange in light and dark by N-limited cells of Chlamydomonas reinhardtii indicated that ammonium assimilation was accompanied by an increase in respiratory carbon flow to provide carbon skeletons for amino acid synthesis. Tricarboxylic acid (TCA) cycle carbon flow was maintained by the oxidation of TCA cycle reductant via the mitochondrial electron transport chain. In wild-type cells, inhibitor studies and ¹⁸O₂ discrimination experiments indicated that respiratory electron flow was mediated entirely via the cytochrome pathway in both the light and dark, despite a large capacity for the alternative pathway. In a cytochrome oxidase deficient mutant, or in wild-type cells in the presence of cyanide, the alternative pathway could support the increase in TCA cycle carbon flow. These different mechanisms of oxidation of TCA cycle reductant were reflected by the much greater SHAM sensitivity of ammonium assimilation by cytochrome oxidase-deficient cells as compared to wild type.     

菠菜叶绿体的光抑制部位

有氧条件下,叶绿体的光抑制部位不是专一的。强光可使PSⅡ氧化侧、PSⅡ反应中心、PSⅡ还原侧,PSⅡ及类囊体膜透性都有不同程度的破坏。这种非专一性可能与类囊体膜蛋白在强光下的降解有关。无氧条件下,叶绿体的光抑制部位只是在PSⅡ反应中心及Q_B蛋白上。