A rapid method for chloroplast isolation from the green alga Chlamydomonas reinhardtii

This method has been developed to yield highly purified intact chloroplasts from Chlamydomonas reinhardtii. This procedure involves breaking cell-wall-deficient cells by passage through a narrow-bore syringe needle and purifying the intact chloroplasts by differential centrifugation and Percoll gradient centrifugation. This procedure can be completed in less than 3 h and is capable of generating relatively high yields of chloroplasts that should be useful for researchers studying the biochemistry and cell biology of C. reinhardtii chloroplasts.     

Photosynthetic activity of spinach chloroplasts after isopycnic centrifugation in gradients of silica

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 CO₂ and evolved O₂ more rapidly than the crude suspensions; the stripped chloroplasts were inactive. During the photosynthetic fixation of ¹⁴CO₂ 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.     

A New Chloroplast Protein Is Induced by Growth on Low CO(2) in Chlamydomonas reinhardtii

The biosynthesis of a 36 kilodalton polypeptide of Chlamydomonas reinhardtii was induced by photoautotrophic growth on low CO₂. Fractionation studies using the cell-wall-deficient strain of C. reinhardtii, CC-400, showed that this polypeptide was different from the low CO₂-induced periplasmic carbonic anhydrase. In addition, the 36 kilodalton polypeptide was found to be localized in intact chloroplasts isolated from low CO₂-adapting cultures. This protein may, in part, account for the different inorganic carbon uptake characteristics observed in chloroplasts isolated from high and low CO₂-grown C. reinhardtii cells.     

Highly purified intact chloroplasts from mesophyll protoplasts of the C4 plant Digitaria sanguinalis. Inhibition of phosphoglycerate reduction by orthophosphate and by phosphoenolpyruvate

Purified mesophyll protoplasts from the C₄ plant Digitaria sanguinalis were used to prepare intact mesophyll chloroplasts with low cytoplasmic contamination. The procedure involved breakage of protoplasts, differential centrifugation, partition in a dextran-polyethylene glycol two-phase system, and Percoll density gradient centrifugation. The final chloroplast preparation contained about 80% intact chloroplasts with a phosphoenolpyruvate carboxylase contamination of 0.2–1% of the original protoplast activity, corresponding to 1–6 μmol ¹⁴CO₂ fixed/mg Chl h. The purified chloroplasts showed substrate-dependent oxygen evolution in the range of 40–150 μmol substrate reduced/mg Chl h, with phosphoglycerate or oxaloacetate as substrate. Both reactions were stimulated 1.5 fold by pyruvate and further by addition of the other substrate. These measurements indicated that phosphoglycerate reduction was limited by substrate transport across the chloroplast envelope. Without added substrate, the chloroplasts consumed oxygen via pseudo-cyclic electron transport in the light. Also this reaction was stimulated by pyruvate. Phosphoglycerate-dependent oxygen evolution was inhibited by P_i and by phosphoenolpyruvate to about the same extent with purified chloroplasts, but only by P_i with protoplast extracts. This suggests that phosphoglycerate, P_i and phosphoenolpyruvate share a common carrier, similar to the P_i-translocator in C₃ chloroplasts, and that the lack of inhibition obtained with phosphoenolpyruvate and unpurified chloroplasts is artefactual, possibly due to oxaloacetate formation from added phosphoenolpyruvate and concomitant stimulation of oxygen evolution by oxaloacetate reduction. Furthermore, the results suggest that phosphoenolpyruvate is transported with a K_m similar to that of P_i in C₄ mesophyll chloroplasts.     

Cellular Fractionation of Chlamydomonas reinhardii with Emphasis on the Isolation of the Chloroplast

A method for cellular fractionation of Chlamydomonas reinhardii, SAG 11-32/b, and isolation of intact chloroplasts from synchronized cells of the alga is described. The procedure for cell fractionation comprises essentially four steps: (1) protoplast production with autolysine; (2) lysis of the protoplasts with digitonin; (3) aggregation of broken protoplasts; and (4) separation of organelles by differential centrifugations.

Replacing the differential centrifugations (step 4) by Percoll cushion centrifugations yields intact chloroplasts. Starting with 100 milliliters of an algal culture containing 3000 micrograms chlorophyll, intact chloroplasts with 100 to 200 micrograms of chlorophyll can be isolated. Envelope integrity is about 90% (ferricyanide assay). Examination of the chloroplasts by electron microscopy and marker enzyme activities indicated some mitochondrial and cytoplasmic contamination.

     

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.     

Isolation of Intact and Functional Chloroplasts from Mesophyll and Bundle Sheath Protoplasts of the C(4) Plant Panicum miliaceum

A procedure is described for isolating and purifying mesophyll protoplasts and bundle sheath protoplasts of the C₄ plant Panicum miliaceum. Following enzymic digestion of leaf tissue, mesophyll protoplasts and bundle sheath protoplasts are released and purified by density centrifugation. The lower density of mesophyll protoplasts allowed rapid separation of the two protoplast types. Evidence for separation of mesophyll protoplasts and bundle sheath protoplasts (up to 95% purity) is provided from light microscopy (based on size difference in both chloroplasts and protoplasts), levels of marker enzymes in the preparations (i.e. pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase for mesophyll and ribulose-1,5-bisphosphate carboxylase for bundle sheath), and differences in substrate-dependent O₂ evolution by chloroplasts isolated from protoplasts.     

Evidence for Chloroplastic Succinate Dehydrogenase Participating in the Chloroplastic Respiratory and Photosynthetic Electron Transport Chains of Chlamydomonas reinhardtii

A method for isolating intact chloroplasts from Chlamydomonas reinhardtii F-60 was developed from the Klein, Chen, Gibbs, Platt-Aloia procedure ([1983] Plant Physiol 72: 481-487). Protoplasts, generated by treatment with autolysine, were lysed with a solution of digitonin and fractionated on Percoll step gradients. The chloroplasts were assessed to be 90% intact (ferricyanide assay) and free from cytoplasmic contamination (NADP isocitrate dehydrogenase activity) and to range from 2 to 5% in mitochondrial contamination (cytochrome c oxidase activity). About 25% of the cellular succinate dehydrogenase activity (21.6 micromoles per milligram chlorophyll per hour, as determined enzymically) was placed within the chloroplast. Chloroplastic succinate dehydrogenase had a K_m for succinate of 0.55 millimolar and was associated with the thylakoidal material derived from the intact chloroplasts. This same thylakoidal material, with an enzymic assay of 21.6 micromoles per milligram chlorophyll per hour was able to initiate a light-dependent uptake of oxygen at a rate of 16.4 micromoles per milligram chlorophyll per hour when supplied with succinate and methyl viologen. Malonate was an apparent competitive inhibitor of this reaction. The succinate dehydrogenase activity present in the chloroplast was sufficient to account for the photoanaerobic rate of acetate dissimilation in H₂ adapted Chlamydomonas (M Gibbs, RP Gfeller, C Chen [1986] Plant Physiol 82: 160-166).     

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.

     

Effects of O(2) and CO(2) Concentration on the Steady-State Fluorescence Yield of Single Guard Cell Pairs in Intact Leaf Discs of Tradescantia albiflora: Evidence for Rubisco-Mediated CO(2) Fixation and Photorespiration in Guard Cells

A procedure for following changes in the steady-state yield of chlorophyll a fluorescence (F_s) from single guard cell pairs in variegated leaves of Tradescantia albiflora is described. As an indicator of photosynthetic electron transport, F_s is a very sensitive indirect measure of the balance of adenosine 5′-triphosphate (ATP) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), producing reactions with the sink reactions that utilize those light-generated products. We found that F_s under constant light is sensitive to manipulation of ambient CO₂ concentrations, as would be expected if either phosphoenolpyruvate carboxylase or ribulose-1, 5 bisphosphate carboxylase/oxygenase (Rubisco)-dependent CO₂ fixation is the sink for photosynthetic ATP and NADPH in guard cells. However, we also found that changing O₂ concentration had a strong effect on fluorescence yield, and that O₂ sensitivity was only evident when the concentration of CO₂ was low. This finding provides evidence that both O₂ and CO₂ can serve as sinks for ATP and NADPH produced by photosynthetic electron transport in guard cell chloroplasts. Identical responses were observed with mesophyll cell chloroplasts in intact leaves. This finding is difficult to reconcile with the view that guard cell chloroplasts have fundamentally different pathways of photosynthetic metabolism from other chloroplasts in C₃ plants. Indeed, Rubisco has been detected at low levels in guard cell chloroplasts, and our studies indicate that it is active in the pathways for photosynthetic carbon reduction and photorespiration in guard cells.     

Preparation of chlamydomonas chloroplasts for the in vitro import of polypeptide precursors

To study the import of polypeptide precursors we have adapted and compared two procedures for the isolation of competent chloroplasts from the green unicellular alga, Chlamydomonas reinhardtii: silicasol gradient centrifugation and elutriation. The chloroplasts actively import the precursor of the small subunit of ribulose bisphosphate carboxylase-oxygenase in vitro.     

Isolation of Bundle Sheath Cell Chloroplasts from the NADP-ME Type C(4) Plant Zea mays: Capacities for CO(2) Assimilation and Malate Decarboxylation

Bundle sheath chloroplasts have been isolated from Zea mays leaves by a procedure involving enzymic digestion of mechanically prepared strands of bundle sheath cells followed by gentle breakage and filtration. The resulting crude chloroplast preparation was enriched by Percoll density layer centrifugation to yield intact chloroplasts (about 20 micrograms chlorophyll per 10-gram leaf tissue) with high metabolic activities. Based on activities of marker enzymes in the chloroplast and bundle sheath cell extracts, the chloroplasts were essentially free of contamination by other organelles and cytoplasmic material, and were generally about 70% intact. Chlorophyll a/b ratios were high (about 10). With appropriate substrates these chloroplasts displayed high rates of malate decarboxylation, measured as pyruvate formation, and CO₂ assimilation (maximum rates approximately 5 and 3 micromoles per minute per milligram chlorophyll, respectively). These activities were light dependent, linear for at least 20 minutes at 30°C, and displayed highest rates at pH 8.0. High metabolic rates were dependent on addition of an exogenous source of carbon to the photosynthetic carbon reduction cycle (3-phosphoglycerate or dihydroxyacetone phosphate) and a nucleotide (ATP, ADP, or AMP), as well as aspartate. Generally, neither malate decarboxylation nor CO₂ assimilation occurred substantially in the absence of the other activity indicating a close relationship between these processes. Presumably, NADPH required for the photosynthetic carbon reduction cycle is largely supplied during the decarboxylation of malate by NADP-malic enzyme. The results are discussed in relation to the role of bundle sheath chloroplasts in C₄ photosynthesis by species of the NADP-malic enzyme type.     

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.     

Orientation of photosystem-I pigments. Investigation by low-temperature linear dichroism and polarized fluorescence emission

Using absorption and fluorescence experiments at low temperature with polarized light on oriented samples, the orientation of PS-I-related pigments, both in green plants and in Chlamydomonas reinhardtii, has been investigated on isolated pigment-protein complexes and intact thylakoids. The following observations have been made. (i) The isolation procedure of PS I₁₁₀, PS I₆₅, LHC I and CP0) particles from pea and C. reinhardtii do not alter significantly the intrinsic orientation of the pigments inside the complexes; (ii) Chl b is a structural component of PS I, linked to the peripheral antenna, with an orientation with respect to the thylakoid plane different from that observed in the main light-harvesting complex (iii) PS I₆₅ (i.e., ‘core’ PS I) of pea and C. reinhardtii contains identical chromophores having the same orientation with respect to the geometrical longest axis (axes) of the complexes. (iv) LHC I and CP0 (i.e., PS I ‘peripheral antenna’) of pea and C. reinhardtii have identical oriented chromophores, except that a long-wavelength component with a high anisotropy is only present in green plants. This set of pigments, which absorbs at 705–725 nm, has the same orientation as the dipoles emitting F₇₃₅ and also as the Q_Y transition of P-700. (v) All the long-wavelength fluorescence properties of the various studied membranes are explained by these data on isolated PS I complexes: wild-type C. reinhardtii and Chl-b-less barely fluoresce from the core pigments, while a CP1 deficient mutant of C. reinhardtii and wild-type barley fluoresce from the antenna pigments.     

Alpha-ketoglutarate supply for amino Acid synthesis in higher plant chloroplasts: intrachloroplastic localization of NADP-specific isocitrate dehydrogenase

Isocitrate dehydrogenase was found in Pisum sativum chloroplasts purified on sucrose density gradients. A chloroplast-enriched pellet obtained by differential centrifugation formed two chlorophyll-containing bands. The lower one containing intact chloroplasts had NADP-specific isocitrate dehydrogenase and triose-phosphate isomerase activities. Mitochondria and peroxisomes were observed to band well away from the intact chloroplast region, as indicated by peak activities of fumarase and catalase, respectively. The presence of isocitrate dehydrogenase in chloroplasts suggests that chloroplasts may generate at least some of the α-ketoglutarate required for glutamate synthesis.     

Free flow electrophoresis of chloroplasts

Highly purified intact chloroplasts were isolated from spinach (Spinacia oleracea L.) leaves by free flow electrophoresis. Morphological and biochemical studies showed that the fraction enriched in intact chloroplasts has a higher protein to chlorophyll ratio and a higher linolenic acid content than the broken organelles of the other fraction. The intact chloroplasts prepared by electrophoresis retained their capacity for CO₂ fixation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that this fraction was rich in stroma and lamellae proteins. Free flow electrophoresis, which separates organelles and molecules according to their surface charges, is a good technique for producing purified chloroplasts with complete physiological activities.     

Isolation of dividing chloroplasts with intact plastid-dividing rings from a synchronous culture of the unicellular red alga Cyanidioschyzon merolae

In order to obtain a three-dimensional view of the plastid-dividing ring (PD ring) and promote the biochemical study of plastid division, we developed a procedure to isolate structurally intact dividing chloroplasts (rhodoplasts) possessing PD rings from a highly synchronized culture of the unicellular red alga Cyanidioschyzon merolae. The procedure consists of five steps. (1) The chloroplast division cycle is synchronized by light/dark cycles and treatment with 5-fluorodeoxyuridine. (2) The synchronized cells are treated with hypotonic solution. (3) The swollen cells are lysed in a French Pressure Cell. (4) The lysate is treated with DNase I. (5) The intact chloroplasts are separated by density-gradient centrifugation. The PD ring was visualized by fluorescence microscopy, after labeling the surface proteins of isolated chloroplasts with N-hydroxy-sulfo-succinimidyl biotin and detecting them with fluorescein isothiocyanate avidin. Scanning electron microscopy (SEM) showed that the outer envelopes and PD rings were conserved on the isolated dividing chloroplasts. These are the first fluorescence microscopic and SEM images of the PD ring and they clearly show PD rings encircling isolated dividing chloroplasts in three dimensions. Received: 15 April 1999 / Accepted: 12 May 1999     

Formation of the isocyclic ring of chlorophyll by isolated Chlamydomonas reinhardtii chloroplasts

Chlamydomonas reinhardtii chloroplasts catalyzed two sequential steps of Chl biosynthesis, S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase and Mg-protoporphyrin IX monomethyl ester oxidative cyclase. A double mutant strain of C. reinhardtii was constructed which has a cell wall deficiency and is unable to form chlorophyll in the dark. Dark-grown cells were disrupted with a BioNeb nebulizer under conditions which lysed the plasma membrane but not the chloroplast envelope. Chloroplasts were purified by Percoll density gradient centrifugation. The purified chloroplasts were used to define components required for the biosynthesis of Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) from Mg-protoporphyrin IX. Product formation requires the addition of Mg-protoporphyrin IX, the substrate for S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase which produces Mg-protoporphyrin IX monomethyl ester. The Mg-protoporphyrin IX monomethyl ester that is generated in situ is the substrate for Mg-protoporphyrin IX monomethyl ester oxidative cyclase. The reaction product was identified as Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) by excitation and emission spectrofluorometry and HPLC on ion-paired reverse-phase and polyethylene columns. Mg-2,4-divinylpheoporphyrin a ₅ formation by the coupled enzyme system required O₂ and was stimulated by the addition of NADP⁺, an NADPH regenerating system, and S-adenosyl-l-methionine. Product was formed at a relatively steady rate for at least 60 min.Abbreviations MgDVP Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) - SAM S-adenosyl-l-methionine     

Preparation of chloroplasts from euglena highly active in protein synthesis

Chloroplasts can be obtained by gentle lysis or mild shear of spheroplasts of vitamin B₁₂-deficient Euglena gracilis and then purified by isopycnic sedimentation on gradients of Ludox AM or Percoll. The chloroplasts appear compact and highly refractile by phase contrast or Hoffmann contrast microscopy. Upon incubation with [³H]leucine or [³⁵S]methionine, the chloroplasts incorporate the amino acids into protein at rates that are 100-fold faster than we had previously observed with Euglena and up to 8-fold faster than with chloroplasts of spinach. Euglena chloroplasts prepared by the current procedure are thus qualitatively superior to those previously available from Euglena and at least as active in protein synthesis as chloroplasts from higher plants.     

Fluorescence Properties of Guard Cell Chloroplasts: EVIDENCE FOR LINEAR ELECTRON TRANSPORT AND LIGHT-HARVESTING PIGMENTS OF PHOTOSYSTEMS I AND II

The presence of chloroplasts in guard cells from leaf epidermis, coleoptile, flowers, and albino portions of variegated leaves was established by incident fluorescence microscopy, thus confirming the notion that guard cell chloroplasts are remarkably conserved. Room temperature emission spectra from a few chloroplasts in a single guard cell of Vicia faba showed one major peak at around 683 nanometers. Low-temperature (77 K) emission spectra from peels of albino portions of Chlorophytum comosum leaves and from mesophyll chloroplasts of green parts of the same leaves showed major peaks at around 687 and 733 nanometers, peaks usually attributed to photosystem II and photosystem I pigment systems, respectively. Spectra of peels of V. faba leaves showed similar peaks. However, fluorescence microscopy revealed that the Vicia peels, as well as those from Allium cepa and Tulipa sp., were contaminated with non-guard cell chloroplasts which were practically undetectable under bright field illumination. These observations pose restrictions on the use of epidermal peels as a source of isolated guard cell chloroplasts. Studies on the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive variable fluorescence kinetics of uncontaminated epidermal peels of C. comosum indicated that guard cell chloroplasts operate a normal, photosystem II-dependent, linear electron transport. The above properties in combination with their reported inability to fix CO₂ photosynthetically may render the guard cell chloroplasts optimally suited to supply the reducing and high-energy phosphate equivalents needed to sustain active ion transport during stomatal opening in daylight.