Isolation Of Intact Chloroplasts Research Articles

Purification of Chloroplast Envelope, Thylakoids, and Stroma from Angiosperm Leaves.

Plant cell chloroplasts are bounded by a two-membrane envelope. Their photosynthetic function is based on the development of an operational large internal membrane network, called the thylakoids, and on enzymatic processes present in the chloroplast matrix, called the stroma. Thylakoid membranes are distinct from the chloroplast envelope, and their biogenesis is dependent on biosynthetic and transport activities specific of the chloroplast envelope. Starting with the isolation of intact chloroplasts, the method presents the separation by differential centrifugation of the three compartments. A protocol is detailed for leaves of spinach, Arabidopsis or pea.

Impact of drought stress on physiological characteristics and isolation of chloroplasts in common bean (Phaseolus vulgaris L.)

Chloroplasts are involved in many cellular processes and play an important role in plant stress response. In this study, the effect of drought on different physiological characteristics and the isolation of chloroplasts from leaves of drought-stressed and control plants of common bean (Phaseolus vulgaris L.) are reported. Two cultivars differing in the response to drought were analysed-Tiber and more sensitive Starozagorski eern. The results of relative water content showed that plants were under mild stress conditions after six days without watering. Negative effects of drought stress on the photosynthetic rate and chlorophyll fluorescence parameters of both cultivars were observed. The yield of isolated chloroplasts in control samples of both cultivars was at least three times higher compared to drought-stressed samples. The intactness of the isolated chloroplasts was checked and used for protein isolation. The results of the study suggested that even mild drought stress strongly affected the isolation of intact chloroplasts. Still, intact chloroplasts were enriched and were suitable for downstream proteomic analysis.

Purification of Chloroplasts and Chloroplast Subfractions: Envelope, Thylakoids, and Stroma-From Spinach, Pea, and Arabidopsis thaliana.

Chloroplasts are specific organelles of plant cells dedicated to photosynthesis and delimited by a two-membrane chloroplast envelope. Their photosynthetic function is based on the development of an operational large internal membrane network, called the thylakoids, and on enzymatic processes present in the chloroplast matrix, called the stroma. Thylakoid membranes are clearly different from the chloroplast envelope and their biogenesis is dependent on biosynthetic and transport activities specific of the chloroplast envelope. Starting with the isolation of intact chloroplasts, the method presents the separation by differential centrifugation of the three main compartments of the chloroplast: the stroma, the thylakoids, and the chloroplast envelope. Three different protocols are provided, adapted for starting leaves of spinach, Arabidopsis, and pea.

Isolation and Suborganellar Fractionation of Arabidopsis Chloroplasts.

Chloroplasts are structurally complex organelles containing ~2000-3000 proteins. They are delimited by a double membrane system or envelope, have an inner aqueous compartment called the stroma, and possess a second internal membrane system called the thylakoids. Thus, determining the suborganellar location of a chloroplast protein is vital to understanding or verifying its function. One way in which protein localization can be addressed is through fractionation. Here we present two rapid and simple methods that may be applied sequentially on the same day: (a) The isolation of intact chloroplasts from Arabidopsis thaliana plants that may be used directly (e.g., for functional studies such as protein import analysis), or for further processing as follows; (b) separation of isolated chloroplasts into three suborganellar fractions (envelope membranes, a soluble fraction containing stromal proteins, and the thylakoids). These methods are routinely used in our laboratory, and they provide a good yield of isolated chloroplasts and suborganellar fractions that can be used for various downstream applications.

Glutamine synthetase isoforms in Trientalis europaea: a biochemical and molecular approach

Ion-exchange chromatography of extracts from Trientalis europaea L. leaf tissue have been shown to contain two distinct isoforms of glutamine synthetase (GS). However, analysis by Western blotting has shown that the first peak to elute contains a mixture of large and small GS subunits, whilst the second peak is comprised entirely of a smaller subunit. This is contrary to the widespread assumptions concerning plant GS biochemistry. Isolation of intact chloroplasts and subsequent extraction of GS, followed by ion-exchange chromatography, has shown that the first peak to elute contains a large subunit, and the second chloroplastic peak is composed entirely of the small subunit. This smaller subunit may be present due to it being encoded by a separate chloroplastic GS gene, or it may be present as a product of post-translational modification. DNA sequencing has been used to try and determine which of these may be occurring. The three partial DNA sequences (505 nucleotides) we have obtained from T. europaea have been compared with 64 other sequences available on the NCBI database, which have mainly been obtained from crop species. Neighbour joining and parsimony analysis (1000 bootstrap) has shown support (∼30%) for the separation of plant GS from all other phyla. Within the plant phylum, there is total support for the separation of chloroplastic and cytosolic GS (100%), whilst the cytosolic sequences divide further into monocot and dicot species (77% support by NJ). Further subgroups of plants from the same families is also suggested. This is consistent with previous work containing fewer, but longer (∼1000 nucleotides) GS sequences. The addition of GS sequences obtained from wild plant species, such as T. europaea, to the large amount of information already available on the database, will permit a better understanding of the evolution of this important enzyme.

Jasmonate‐induced lipoxygenase forms are localized in chloroplasts of barley leaves (Hordeum vulgare cv. Salome)

Barley leaves respond to application of (−)‐jasmonic acid (JA), or its methylester (JM) with the synthesis of abundant proteins, so‐called jasmonate induced proteins (JIPs). Here Western blot analysis is used to show a remarkable increase upon JM treatment of a 100 kDa lipoxygenase (LOX), and the appearance of two new LOX forms of 98 and 92 kDa. The temporal increase of LOX‐100 protein upon JM treatment was clearly distinguishable from the additionally detectable LOX forms. JM‐induced LOX forms in barley leaves were compared with those of Arabidopsis and soybean leaves. Both dicot species showed a similar increase of one LOX upon JM induction, whereas, leaves from soybean responded with additional synthesis of a newly formed LOX of 94 kDa.Using immunofluorescence analysis and isolation of intact chloroplasts, it is demonstrated that JM‐induced LOX forms of barley leaves are exclusively located in the chloroplasts of all chloroplast‐containing cells. Analogous experiments carried out with Arabidopsis and soybean revealed a similar plastidic location of JM‐induced LOX forms in Arabidopsis but a different situation for soybean. In untreated soybean leaves the LOX protein was mainly restricted to vacuoles of paraveinal mesophyll cells. Additionally, LOX forms could be detected in cytoplasm and nuclei of bundle sheath cells. Upon JM treatment cytosolic LOX was detectable in spongy mesophyll cells, too. The intracellular location of JM‐induced LOX is discussed in terms of stress‐related phenomena mediated by JM.

Induction of intracellular carbonic anhydrases during the adaptation to low inorganic carbon concentrations in wild-type and ca-1 mutant cells of Chlamydomonas reinhardtii

Mass-spectrometric measurements of 18O exchange from 13C18O2 were used to follow changes in the intracellular carbonic anhydrase (CA) activity of cells of Chlamydomonas reinhardtii Dang, wild type and the ca-1 mutant during adaptation to air. With intact cells as well as with crude homogenates total intracellular CA activity in wild-type cells increased six to tenfold within 4 h after transferring cells from 5% CO2 (high inorganic carbon, Ci) to ambient air (air adapted). After that time the activity slowly declined to a level similar to that observed with cells which had been continuously grown in air (low-Ci grown). In the ca-1 mutant, total CA was induced to a similar extent during 4 h of adaptation; however, absolute activities were two to three times lower in ca-1 than in the wild type regardless of the CO2 supply. When crude extracts from wild-type cells were separated into soluble and insoluble fractions, each fraction contained about half of the internal CA activity. Within 4 h of adaptation, both forms of CA activity were simultaneously enhanced by nine to tenfold, reaching levels similar to those found in low-Cigrown cells. In contrast, in the ca-1 mutant the soluble CA activity was only enhanced by about eightfold while the level of insoluble CA was very low even in low-Ci cells. After isolation of intact chloroplasts from wild-type cells and further subfractionation, around 70–80% of total chloroplastic CA activity was found to be in the insoluble fraction while 17–20% remained in the soluble fraction. Both chloroplastic CA activities were inducible within the first 4 h of adaptation to air, with each of them being eight to ten times higher than in high-Ci algae. After that time their activities were similar to the corresponding CA values in low-Ci-grown cells. In contrast, plastids from high-Ci cells of the ca-1 mutant showed 40% less insoluble-CA activity compared to the wild type and this insoluble-CA activity was not increased at all by transferring algae to air. In addition, no soluble-CA activity was detected in chloroplasts from high-Ci and air-adapted ca-1 cells. These results indicate the presence of three intracellular CA activities in high-Ci air-adapted and low-Ci cells of the wild type and that two of them are associated with the chloroplasts. All three activities are completely induced within the first 4 h of adaptation to air in wild-type cells. In contrast, it was not possible to induce any of the chloroplastic CA activities in the ca-1 mutant. The possibility that the soluble chloroplastic CA represents a pyrenoid-located CA is discussed.

The isolation of intact chloroplasts.

AbstractThe isolation of intact chloroplasts from plant tissue allows the study of processes carried out in this subcellular compartment. Such processes may be enzymatic, e.g., photosynthetic oxygen-evolution, or mechanistic, like the import and targeting of precursor proteins synthesized in the cytosol. The latter is important in the case of the plant molecular biologist wishing to study the subcellular localization of a given protein.KeywordsOxygen EvolutionMuslin ClothChloroplast EnvelopeIntact ChloroplastElectrode ChamberThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Induction, localization and metal content of hydrogenase in the green alga Chlamydomonas reinhardtii.

The hydrogenase enzyme occurring in Chlamydomonas reinhardtii is induced by anaerobic adaptation of the cells. In aerobically growing cells, antibodies against the hydrogenase failed to detect either active or inactive enzyme. However, already 10 min after the onset of anaerobic adaptation, the protein could be detected. The maximal amount of enzyme was reached after 2-3 hours anaerobiosis. Addition of nickel or iron to the growth medium did not influence activity. In atomic absorption experiments, a Ni/Fe ratio of about 1:250 was measured. We, therefore, propose the hydrogenase from C. reinhardtii to be of the Fe-only type. Adaptation in the presence of uncouplers of phosphorylation showed this process to be energy-dependent. From protein synthesis inhibition experiments, it is concluded that the protein is synthesized on cytoplasmic ribosomes and, therefore, must be nuclear encoded. After isolation of intact chloroplasts from adapted cells, the active enzyme was shown, by Western-blotting analysis, to be located in the chloroplasts.

Net fluxes and pools of nitrogenous compounds during suspension culture of photoautotrophicChenopodium rubrumcells

Abstract.Suspension cultured cells ofChenopodium rubrumwere grown photoautotrophically under a diurnal light‐dark cycle of 16‐8h. The following phases of the batch culture were differentiated: a short lag, a cell division phase terminated by a pronounced transition to stationary maintenance which finally gradually passed into senescence. Nitrogen fluxes typical of these stages were followed by measuring uptake of NO3and NH4+from the medium and their incorporation into the cellular fractions of nitrogenous compounds. Activities of seven N‐metabolizing enzymes were determined. Compartmentation of enzymes and nitrogenous compounds was analysed after isolation of intact chloroplasts and vacuoles from protoplasts. Eighty‐two per cent of the N originally present in the medium was taken up and incorporated to an extent of 80% into protein until the end of the division phase. Net protein synthesis ceased upon transition to the stationary phase. During the division phase a vacuolar pool of NO3was established and then maintained throughout the resting phase. Free cellular NH4+was not localized within the vacuole and responded to the ammonium content of the medium. Amino acids accumulated in the cells especially during the stationary phase, during which they were present in the vacuole. Typical nitrogen relations are portrayed as flux diagrams for one day of each of the essential developmental phases. The enzyme activities were easily sufficient to account for the observed flow rates of the corresponding nitrogenous compounds. Hence, uptake of NO3and NH4+must be considered as steps limiting N metabolism inChenopodium rubrumcell suspensions.

Inhibition of the phosphate transporter during isolation of intact chloroplasts from leaves of sunflower

Chloroplasts isolated from spinach leaves by the mechanical method were intact and exhibited high rates of CO2-dependent oxygen evolution whereas chloroplasts isolated from sunflower leaves by the same technique were also intact but showed only low rates of oxygen evolution. The rate of uptake of orthophosphate (Pi) from the suspending medium with sunflower chloroplasts was less than 20% of that in spinach chloroplasts. The apparent Km for Pi transport was lower in sunflower chloroplasts but uptake was competitively inhibited by 3-phosphoglycerate in chloroplasts from both species. Uptake of malate (via the dicarboxylate transporter) and of ATP (via the adenine nucleotide transporter) was also reduced in sunflower chloroplasts compared to spinach chloroplasts. The endogenous Pi content and total exchangeable phosphate pool of sunflower chloroplasts were less than half that in spinach chloroplasts.Addition of a number of possible protective agents to the grinding medium failed to prevent the loss of photosynthetic activity during mechanical isolation of sunflower chloroplasts. Grinding mixtures of spinach and sunflower leaves together indicated that spinach chloroplasts were not inhibited by the sunflower leaf extract. Chloroplasts isolated from sunflower leaves via protoplasts had high rates of CO2-dependent oxygen evolution. The Vmax and Km for Pi uptake, endogenous Pi content and total exchangeable phosphate pool of chloroplasts isolated from sunflower protoplasts were all similar to spinach chloroplasts. It is concluded that inner envelope membrane proteins are damaged during mechanical isolation of sunflower chloroplasts. The decrease in activity of the phosphate transporter and loss of endogenous phosphate may contribute to the low rates of photosynthesis observed in chloroplasts isolated by the mechanical method from leaves of sunflower and possibly other species.

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.

Isolation of intact chloroplasts with high CO2 fixation capacity from sugarbeet leaves containing calcium oxalate

Intact chloroplasts were isolated from sugarbeet leaves by the mechanical disruption technique normally used for spinach. The chloroplast pellet contained a ring of white irregularly shaped crystals which were identified as calcium oxalate. The chloroplasts were greater than 90% intact yet good rates of CO2 fixation were only obtained when inorganic pyrophosphate or 3-phosphoglycerate were added to the assay medium. Chloroplasts free of calcium oxalate were prepared by purification on a three step Percoll gradient. These purified chloroplasts were highly intact and showed high rates of CO2 fixation without adding inorganic pyrophosphate or 3-phosphoglycerate. With optimal assay conditions (0.2 mM orthophosphate and pH 8.0) rates of 110-130 μ mole per milligram chlorophyll per hour were routinely obtained. It is concluded that intact chloroplasts capable of high rates of CO2 fixation can be prepared from sugarbeet leaves using a simple three step Percoll gradient.

Isolation of intact chloroplasts with high CO2 fixation capacity from sugarbeet leaves containing calcium oxalate

Intact chloroplasts were isolated from sugarbeet leaves by the mechanical disruption technique normally used for spinach. The chloroplast pellet contained a ring of white irregularly shaped crystals which were identified as calcium oxalate. The chloroplasts were greater than 90% intact yet good rates of CO2 fixation were only obtained when inorganic pyrophosphate or 3-phosphoglycerate were added to the assay medium. Chloroplasts free of calcium oxalate were prepared by purification on a three step Percoll gradient. These purified chloroplasts were highly intact and showed high rates of CO2 fixation without adding inorganic pyrophosphate or 3-phosphoglycerate. With optimal assay conditions (0.2 mM orthophosphate and pH 8.0) rates of 110-130 μ mole per milligram chlorophyll per hour were routinely obtained. It is concluded that intact chloroplasts capable of high rates of CO2 fixation can be prepared from sugarbeet leaves using a simple three step Percoll gradient.

Isolation of intact chloroplasts from spinach leaf by centrifugation in gradients of the modified silica "Percoll".

The isolation of the photosynthetically competent chloroplast preparations was undertaken by means of the density gradient centrifugation on the modified silica sol “Percoll.” A clear separation of...

Isolation of Intact Chloroplasts and Other Cell Organelles from Spinach Leaf Protoplasts

Freshly prepared spinach leaf protoplasts were gently ruptured by mechanical shearing followed by sucrose density gradient centrifugation to separate constituent cell organelles. The isolation of intact Class I chloroplasts (d = 1.21) in high yield, well separated from peroxisomes and mitochondria, was evidenced by the specific localization of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), NADP triose-P dehydrogenase (EC 1.2.1.9), and carbonic anhydrase (EC 4.2.1.1) in the fractions. A clear separation of chloroplastic ribosomes from the soluble cytoplasmic ribosomes was also demonstrated by the band patterns of constituent RNA species in the polyacrylamide gel electrophoresis. Localization of several enzyme activities specific to leaf peroxisomes, e.g. catalase (EC 1.11.1.6), glycolate oxidase (EC 1.1.3.1), glyoxylate reductase (EC 1.1.1.26), glutamate glyoxylate aminotransferase (EC 2.6.1.4), serine glyoxylate aminotransferase, and alanine glyoxylate aminotransferase (EC 2.6.1.12) in the peroxisomal fractions (d = 1.25), was demonstrated. Overall results show the feasibility of the method for the isolation of pure organelle components in leaf tissues.

Isolation of Intact Chloroplasts from Euglena gracilis by Zonal Centrifugation

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.

Glutaraldehyde as an effective stabilizer for the isolation of intact chloroplasts<xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

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