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1.
Simplified Procedure for the Isolation of Intact Chloroplasts from Chlamydomonas reinhardtii 总被引:6,自引:4,他引:2
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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. 相似文献
2.
Chloroplasts from spinach (Spinacia oleracea L.) can be resolved by isopycnic centrifugation in density gradients of the silica sol Ludox AM into two zones, one containing the intact and the other the stripped Chloroplasts. Factors affecting the separation and the photosynthetic activity of the intact chloroplasts recovered from the gradients were investigated and a standard set of conditions proposed. The anomalous influence of polyethylene glycols on the banding densities of both stripped and intact chloroplasts was studied and the observed “density shifts” discussed in terms of their possible cause and significance. The general method was scaled up by means of continuous-flow zonal centrifugation to prepare intact chloroplasts in amounts corresponding to tens of milligrams of chlorophyll. 相似文献
3.
Counter-current distribution in an aqueous Dextran-polyethylene glycol two-phase system has been used to fractionate membrane fragments obtained by press treatment of Class II chloroplasts. By the counter-current distribution technique membrane particles are separated according to their surface properties such as charge and hydrophobicity.The fractions obtained were analysed with respect to photochemical activities, chlorophyll and P-700 contents. The Photosystem II enrichment after counter-current distribution was better than that obtained by differential centrifugation of the disrupted chloroplasts. However, the best separation of Photosystem I and II enriched particles could be achieved if differential centrifugation was combined with the counter-current distribution technique.Each centrifugal fraction could be further separated into Photosystems I and II enriched fractions since the Photosystem II particles preferred the dextran-rich bottom phase while the Photosystem I particles preferred the polyethylene glycol-rich top phase. By this procedure it was possible, without the use of detergents, to obtain vesicles which were more enriched in Photosystem II as compared to intact grana stacks.The partition behaviour of undisrupted Class II chloroplasts and the Photosystem I centrifugal fraction was the same. This similarity indicates that the membrane which is exposed to the surrounding polymers by the Class II chloroplasts is the Photosystem I rich membrane of the stroma lamellae. 相似文献
4.
Counter-current distribution in an aqueous Dextran-polyethylene glycol two-phase system has been used to fractionate membrane fragments obtained by press treatment of Class II chloroplasts. By the counter-current distribution technique membrane particles are separated according to their surface properties such as charge and hydrophobicity. The fractions obtained were analysed with respect to photochemical activities, chlorophyll and P-700 contents. The Photosystem II enrichment after counter-current distribution was better than that obtained by differential centrifugation of the disrupted chloroplasts. However, the best separation of Photosystem I and II enriched particles could be achieved if differential centrifugation was combined with the counter-current distribution technique. Each centrifugal fraction could be further separated into Photosystems I and II enriched fractions since the Photosystem II particles preferred the dextran-rich bottom phase while the Photosystem I particles preferred the polyethylene glycol-rich top phase. By this procedure it was possible, without the use of detergents, to obtain vesicles which were more enriched in Photosystem II as compared to intact grana stacks. The partition behaviour of undisrupted Class II chloroplasts and the Photosystem I centrifugal fraction was the same. This similarity indicated that the membrane which is exposed to the surrounding polymers by the Class II chloroplasts is the Photosystem I rich membrane of the stroma lamellae. 相似文献
5.
Photosynthetic activity of spinach chloroplasts after isopycnic centrifugation in gradients of silica 总被引:16,自引:8,他引:8
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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. 相似文献
6.
Purity of Chloroplasts Prepared from the Siphonous Green Alga, Caulerpa simpliciuscula, as Determined by Their Ultrastructure and Their Enzymic Content 总被引:1,自引:1,他引:0
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The ultrastructure and enzyme distribution in chloroplasts and other subcellular fractions isolated from the siphonous green alga, Caulerpa simpliciuscula, are described. The isolated chloroplasts were similar in appearance to those in the tissue from which they were derived, and in typical preparations 70% or more were intact. Chloroplasts which had lost their outer envelopes could be separated from intact plastids by centrifugation at low speeds through gradients of colloidal silica. Intact chloroplasts separated in this way retained their photosynthetic capacity and were impermeable to ferricyanide ions. The chloroplast preparations separated by differential centrifugation and refractionated using either discontinuous or continuous Percoll gradients contained non-chloroplast material. It was estimated that this amounted to a maximum of 10% of the mitochondrial population and 6% of cytoplasm extracted from the plant. The contaminating material surrounded the chloroplasts in a thin layer and was surrounded by a membrane. 相似文献
7.
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 Pi and by phosphoenolpyruvate to about the same extent with purified chloroplasts, but only by Pi with protoplast extracts. This suggests that phosphoglycerate, Pi and phosphoenolpyruvate share a common carrier, similar to the Pi-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 Km similar to that of Pi in C4 mesophyll chloroplasts. 相似文献
8.
Shin-ya Miyagishima Ryuuichi Itoh Saburo Aita Haruko Kuroiwa Tsuneyoshi Kuroiwa 《Planta》1999,209(3):371-375
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 相似文献
9.
10.
Intact protoplasts are ruptured by rapid centrifugation through a narrow-aperture nylon mesh and the intact chloroplasts are then separated from the cytoplasm by sedimentation through a layer of silicone oil below the mesh. Within 6 to 8 s of starting the centrifuge, 90% of the chloroplasts are separated into the pellet fraction which contains only 10 to 15% contamination by mitochondria and peroxisomes and less than 5% contamination by soluble cytoplasm as judged by the distribution of marker enzymes. This technique should allow determination of the distribution of metabolites between the chloroplast and cytoplasmic compartments of intact protoplasts. 相似文献
11.
The subcellular distribution of pyruvate-degrading enzymes has been determined in Chlamydomonas reinhardtii (Dangeard) by protoplast induction with autolysine, dig-itonin lysis and further fractionation by differential centrifugation using a Percoll cushion. Mitochondrial and plastidic fractions contained intact and physiologically competent organelles - RC 1.7, ADP/O 2.7 and rate of malate oxidation 76 nmol O, (mg protein)-1 min-1 for mitochondria, CO2; fixation 46.8 μmol (mg Chi)-1 h-1 for chloroplasts.
Results from protoplast fractionation were further confirmed by the determination of enzyme activities within trypsin-treated organelles. Mitochondria (formate fermentation) and chloroplasts (chlorofermentation) were shown to possess the capacity for anaerobic pyruvate degradation. Pyruvate dehydrogenase (NAD+ , EC 1.2.4.1), pyruvate formate-lyase (EC 2.3.1.54) and lactate dehydrogenase (NADH, EC 1.1.1.27) showed equal distribution between mitochondria and chloroplasts, whereas activities of phosphotransacetylase (EC 2.3.1.8) and acetate kinase (EC 2.7.2.1) were only detectable in the mitochondrial fraction. NADH- and NADPH-dependent activities of both alcohol dehydrogenase (EC 1.1.1.1) and aldehyde dehydrogenase (acylating, EC 1.2.1.10) were localized in the mitochondrial and cytoplasmic or the plastidic and cytoplasmic fractions, respectively, whereas pyruvate decarboxylase (EC 4.1.1.1) was only detected in the cytoplasmic fraction. 相似文献
Results from protoplast fractionation were further confirmed by the determination of enzyme activities within trypsin-treated organelles. Mitochondria (formate fermentation) and chloroplasts (chlorofermentation) were shown to possess the capacity for anaerobic pyruvate degradation. Pyruvate dehydrogenase (NAD
12.
A New Chloroplast Protein Is Induced by Growth on Low CO(2) in Chlamydomonas reinhardtii 总被引:3,自引:3,他引:0
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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. 相似文献
13.
Alpha-ketoglutarate supply for amino Acid synthesis in higher plant chloroplasts: intrachloroplastic localization of NADP-specific isocitrate dehydrogenase 总被引:1,自引:0,他引:1
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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. 相似文献
14.
Isolation of Intact Chloroplasts from Euglena gracilis by Zonal Centrifugation 总被引:8,自引:3,他引:5
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Vasconcelos A Pollack M Mendiola LR Hoffmann HP Brown DH Price CA 《Plant physiology》1971,47(2):217-221
Chloroplasts were separated from Euglena gracilis by zonal centrifugation at low speed in density gradients of Ficoll or dextran. The chloroplasts were intact by the criteria of ultrastructure and their content of ribulose diphosphate carboxylase and soluble protein. The chloroplasts also contained ribosomes and ribosomal RNA uncontaminated by the corresponding cytoplasmic particles. 相似文献
15.
Enzymic determination of metabolites in the subcellular compartments of spinach protoplasts 总被引:29,自引:24,他引:5
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A method for determining the subcellular metabolite levels in spinach protoplasts is described. The protoplasts are disrupted by centrifugation through a nylon net, releasing intact chloroplasts which pass through a layer of silicone oil into perchloric acid while the remaining cytoplasmic components remain over the oil and are simultaneously quenched as acid is centrifuged into them. Cross-contamination is measured and corrected for using ribulose 1,5-bisphosphate as a chloroplastic marker and phosphoenolpyruvate carboxylase as a cytoplasmic marker. A method for separation of intact protoplasts from the medium by silicone oil centrifugation is described, which allows a correction to be made for the effect of free chloroplasts and broken protoplasts. Methods for inhibiting chloroplast photosynthesis, without inhibiting protoplasts, are presented. It is demonstrated that ribulose 1,5-bisphosphate, ATP, ADP, AMP, inorganic phosphate, hexose phosphate, triose phosphate, fructose 1,6-bisphosphate, and 3-phosphoglycerate can be reliably recovered in the subcellular fractions isolated from protoplasts, and measured by enzymic substrate analysis. 相似文献
16.
Slow Passive Diffusion of Orthophosphate between Intact Isolated Chloroplasts and Suspending Medium 总被引:14,自引:7,他引:7
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Isolated spinach chloroplasts purified by isopycnic centrifugation in density gradients of Percoll were found to be highly intact, to be devoid of extrachloroplastic contaminations, and to retain a high rate of CO2-dependent O2 evolution. 相似文献
17.
Isolation of intact spinach chloroplasts in the CF-6 continuous-flow zonal rotor; implications for membrane-bound organelles 总被引:3,自引:0,他引:3
Intact chloroplasts were separated from large volumes of spinach brei by continuous-flow zonal centrifugation in the CF-6 rotor in gradients of colloidal silica. Tests with the Z-15/Mark 10 rotor showed that the external membranes of chloroplasts were sensitive to high rotor speeds and especially to the bore of the fluid seal. Enlargement of the CF-6 seal channel diameter to 4 mm resulted in almost complete recovery of the intact chloroplasts. 相似文献
18.
Franklin S Ngo B Efuet E Mayfield SP 《The Plant journal : for cell and molecular biology》2002,30(6):733-744
Reporter genes have been successfully used in chloroplasts of higher plants, and high levels of recombinant protein expression have been reported. Reporter genes have also been used in the chloroplast of Chlamydomonas reinhardtii, but in most cases the amounts of protein produced appeared to be very low. We hypothesized that the inability to achieve high levels of recombinant protein expression in the C. reinhardtii chloroplast was due to the codon bias seen in the C. reinhardtii chloroplast genome. To test this hypothesis, we synthesized a gene encoding green fluorescent protein (GFP) de novo, optimizing its codon usage to reflect that of major C. reinhardtii chloroplast-encoded proteins. We monitored the accumulation of GFP in C. reinhardtii chloroplasts transformed with the codon-optimized GFP cassette (GFPct), under the control of the C. reinhardtii rbcL 5'- and 3'-UTRs. We compared this expression with the accumulation of GFP in C. reinhardtii transformed with a non-optimized GFP cassette (GFPncb), also under the control of the rbcL 5'- and 3'-UTRs. We demonstrate that C. reinhardtii chloroplasts transformed with the GFPct cassette accumulate approximately 80-fold more GFP than GFPncb-transformed strains. We further demonstrate that expression from the GFPct cassette, under control of the rbcL 5'- and 3'-UTRs, is sufficiently robust to report differences in protein synthesis based on subtle changes in environmental conditions, showing the utility of the GFPct gene as a reporter of C. reinhardtii chloroplast gene expression. 相似文献
19.
Angelina Angelova Sang-Hycuk Park John Kyndt Kevin Fitzsimmons Judith K. Brown 《Journal of applied phycology》2014,26(1):209-218
With the increasing world demand for biofuel, a number of oleaginous algal species are being considered as renewable sources of oil. Chlorella protothecoides Krüger synthesizes triacylglycerols (TAGs) as storage compounds that can be converted into renewable fuel utilizing an anabolic pathway that is poorly understood. The paucity of algal chloroplast genome sequences has been an important constraint to chloroplast transformation and for studying gene expression in TAGs pathways. In this study, the intact chloroplasts were released from algal cells using sonication followed by sucrose gradient centrifugation, resulting in a 2.36-fold enrichment of chloroplasts from C. protothecoides, based on qPCR analysis. The C. protothecoides chloroplast genome (cpDNA) was determined using the Illumina HiSeq 2000 sequencing platform and found to be 84,576 Kb in size (8.57 Kb) in size, with a GC content of 30.8 %. This is the first report of an optimized protocol that uses a sonication step, followed by sucrose gradient centrifugation, to release and enrich intact chloroplasts from a microalga (C. prototheocoides) of sufficient quality to permit chloroplast genome sequencing with high coverage, while minimizing nuclear genome contamination. The approach is expected to guide chloroplast isolation from other oleaginous algal species for a variety of uses that benefit from enrichment of chloroplasts, ranging from biochemical analysis to genomics studies. 相似文献
20.
Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires theintroduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins ormetabolic pathways.In order to accomplish the expression of multiple genes in a single transformationevent,we inserted both large and small subunits of allophycocyanin gene (apcA and apcB) into Chlamydomonasreinhardtii chloroplast expression vector,resulting in papc-S.The constructed vector was then introducedinto the chloroplast of C.reinhardtii by micro-particle bombardment.Polymerase chain reaction and Southernblot analysis revealed that the two genes had integrated into the chloroplast genome.Western blot andenzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria couldbe correctly expressed in the chloroplasts of C.reinhardtii.The expressed foreign protein in transformantsaccounted for about 2%-3% of total soluble proteins.These findings pave the way to the reconstitution ofmulti-subunit proteins or metabolic pathways in transgenic C.reinhardtii chloroplasts in a single transformationevent. 相似文献