Factors affecting the separation of photosynthetically competent chloroplasts in gradients of silica sols

Morgenthaler, J.‐J.; Marsden, Margery P. F.; Price, Colin

doi:10.1016/0003-9861(75)90253-2

Cited by 79 publications

(34 citation statements)

References 21 publications

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“…Chloroplasts prepared by this protocol are separated from mitochondria and nuclei by the initial differential centrifugations and from adherent cellular contaminants such as starch, chromatin, and free membranes during the isopycnic centrifugation. Chloroplasts exposed to purified Ludox retain photosynthetic activity (8,13) and normal ultrastructure (9 (Fig. 3).…”

Section: Resultsmentioning

confidence: 97%

“…Morgenthaler et al (9) have described a variety of silica sol gradients which have proved useful in the separation of "stripped" and "intact" chloroplasts. Under optimal conditions stripped and intact chloroplasts banded at densities of 1.05 g/ml and 1.12 g/ml, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…Figure 3; density was determined by weighing, and refractive index was determined on a Bausch and Lomb refractometer. The properties of Ludox gradients are described in more detail in the publications of Morgenthaler et al (8,9) and Price et al (11) including the phenomenon of "position reversal" of isopycnically separated materials after the addition of various molecules such as PEG, Ficoll, etc. The theoretical basis for the peculiarities of separation in mixed polymer gradients has not been determined.…”

Section: Methodsmentioning

confidence: 99%

“…Mesophyll chloroplasts can be prepared from protoplasts (5), however, a few methods for quickly preparing intact bundle sheath chloroplasts and for separating mixtures of dimorphic chloroplasts have been described. Previous studies demonstrated the utility of silica sol gradients for the preparation of chloroplasts (8,9,11,16) and suggested that suitable centrifugation conditions could be devised to separate effectively dimorphic chloroplasts in a pseudoisopycnic gradient. The method outlined here provides purified chloroplasts suitable for a variety of analytical procedures; although the method yields small quantities of purified organelles using conventional rotors it should be suitable for zonal rotor centrifugation.…”

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confidence: 99%

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Use of Silica Sol Step Gradients to Prepare Bundle Sheath and Mesophyll Chloroplasts from Panicum maximum

Walbot

1977

Plant Physiol.

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The first method for the direct separation of mesophyil and bundle sheath chloroplasts from whole tissue homogenates of a C4 plant is described. Centrifugation of mixed chloroplast preparations from Panicum maximum through low viscosity silica sol gradients effectively separates large, starch-containing chloroplasts from smaller plastids. The large chloroplasts are judged to be bundle sheath chloroplasts on the basis of microscopic appearance, the presence of starch grains, the protein complement displayed on sodium dodecyl sulfate acrylamide gels, and the exdusive localiztion of ribulose bisphosphate carboxylase activity in these plastids. As a measure of intactness both the large (bundle sheath) and small (mesophyll) chloroplasts contain glyceraldehyde-3-phosphate NADP-dependent dehydrogenase activity that is greatly enhanced by plastid lysis and both chloroplast preparations are impermeable to deoxyribonudease. Chloroplast the starch content of the BSC chloroplasts. Optimal separation is achieved when the MC chloroplasts contain no microscopically detectable starch and the BSC chloroplasts contain approximately 10 to 20 starch grains/plastid. Plants to be used as a source of chloroplasts were checked for chloroplast starch content by incubating hand cut 0.5-mm leaf sections in a drop of iodine starch detection reagent for 1 min, rinsing quickly in several drops of 70% ethanol to fix the tissue and remove excess color, and observing the material using Nomarski differential interference contrast optics (10). As shown in Figure 1A, the BSC chloroplasts are clearly seen to contain starch while the MC chloroplasts contain no visible starch. If visible starch was detected in the MC chloroplasts, the plants were kept in the dark for 24 to 36 hr to destarch the leaf and then returned to the light to allow starch accumulation in the BSC chloroplasts.Source and Purification of Reagents. All biochemicals were obtained from Sigma Chemical Co. Ribose-5-P was treated with Chelex 100 as previously described (16) to remove heavy metals that may interfere with the spectrophotometric assay of RuDPCase. Ludox was a gift of E. I. du Pont de Nemours & Co.; of the several grades of Ludox tested, purified HS-40 was found to be least disruptive to chloroplast structure. Ludox was purified as described by Morgenthaler et al. (8) and stored at room temperature. The nominal initial per cent SiO2 was 40%; purified material was 38% SiO2 (w/v) determined by refractometry.Preparation of Chloroplasts. Twenty to 100 g of P. maximum leaves were rinsed for 15 min in cold tap water. The leaf segments were roughly cut with scissors into 1-cm pieces and 20-g samples loaded into a precooled Waring Blendor containing 150 ml of blending medium (0.33 M sorbitol, 5 mM MgCl2, 50 mM tris-HCI (pH 8), 0.1% BSA (fraction V), 0.1 mm mercaptoethanol). Leaves were blended for 15 sec in 2-to 3-sec bursts at full speed; the liquid was filtered sequentially through 64-,um and 20-,um nylon netting; the filtrate was centrifuged at lOOg for 5 min to remo...

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Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Use of Silica Sol Step Gradients to Prepare Bundle Sheath and Mesophyll Chloroplasts from Panicum maximum

Walbot

1977

Plant Physiol.

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“…Leaves were homogenized with a Waring blender three times for 5 s in Nakatani and Barber's medium (15) and intact chloroplasts were isolated by the method of Morgenthaller et al (14) using a Percoll gradient. Aliquots of intact chloroplasts were ruptured by dilution in 50 mM Tris-HCl (pH 6.8), and 5 mM MgCl2, and centrifuged at 12,000g for 15 min to separate the stroma and thylakoid fractions.…”

Section: Percoll Gradient Centrifugationmentioning

confidence: 99%

Senescence-Induced, Thylakoid-Bound Diisopropylfluorophosphate-Binding Protein in Spinach

Kawasaki

Takeuchi²

1989

Plant Physiol.

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Changes in diisopropylfluorophosphate (DFP)-binding proteins during development and senescence of spinach (Spinacia oleracea) leaves were followed using [3H]DFP and sodium dodecylsulfate-polyacrylamide gel electrophoresis-fluorography. Experiments using a series of aging stages of leaves attached to plants and ones with detached leaves stored in the dark both showed that a protein of 38 kilodaltons was the only major DFP-binding protein in the membrane fraction and that its DFP-binding increased markedly as senescence proceeded, corresponding with the degradation of leaf protein. DFP binding to the 38-kilodalton protein was not affected by membrane solubilization with Triton X-100, and gradually decreased upon preservation of the membranes. The DFP binding was inhibited completely by phenylmethane-sulfonyl fluoride and slightly by p-chloromercuribenzoic acid, suggesting a serine protease-like character of the protein and a possible contribution of SH residues to the binding. Both differential and Percoll-gradient centrifugation indicated that the 38-kilodalton protein was localized in thylakoid membranes. The sedimentation behavior of the detergent-solubilized protein indicated that it belongs to a complex different from photosystem 1, photosystem 11, or coupling factor I of the ATP-synthesizing complex.(12, 27, 28), and the decrease in chloroplast content has been shown to be due to increased degradation, rather than to a decrease in synthesis (7, 10).Many attempts have been made to identify the degradative enzymes, in most cases proteases, functioning in the retrieval of nutrients (11,13,18,19), but little success has been achieved. One reason may be that the vacuole's large pool of degradative enzymes, which are not necessarily related to senescence (24), makes it difficult to detect other specific organellar enzymes in cell homogenates. Also, the substrate specificities of these enzymes are sometimes so narrow that a survey of enzymes with the small number of substrates hitherto used would seem to have limited prospects. Therefore, detection of proteases related to senescence may require alternative approaches.DFP3 is known to combine irreversibly with active serine residues of proteases and esterases, and proteins showing DFP binding activity can be analyzed simultaneously with high sensitivity using SDS-PAGE and fluorography (4, 21). In this study, aging-associated changes in DFP-binding proteins in spinach leaves were followed in an attempt to detect senescence-related enzymes. One senescence-induced protein was identified and analyzed further.

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Glutamine synthetase genes of pea encode distinct polypeptides which are differentially expressed in leaves, roots and nodules.

Tingey¹,

Walker²,

Coruzzi³

1987

The EMBO Journal

191

148

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We have characterized the distinct polypeptides, primary translation products and mRNAs encoding glutamine synthetase (GS) in the various organs of pea. Western blot analysis of soluble protein has identified five distinct GS polypeptides which are expressed at different relative levels in leaves, roots and nodules of pea. Of the two GS polypeptides in leaves (44 and 38 kd), the 44‐kd GS polypeptide is predominant and is localized to the chloroplast stroma. In roots, the predominant GS polypeptide is 38 kd. Upon Rhizobium infection of roots, three 37‐kd GS polypeptides increase in abundance in the nodules relative to uninfected roots. cDNA clones encoding three different GS mRNAs have been characterized. Hybrid‐select translation has identified three different GS primary translation products (49, 38 and 37 kd). Two cDNA clones (pGS134 and pGS341) are homologous to GS mRNAs most abundant in nodules which encode the 38‐ and 37‐kd GS primary translation products. A third cDNA (pGS197) corresponds to a larger GS mRNA species specific to leaf poly(A) RNA, which encodes a 49‐kd putative precursor to the mature chloroplast GS polypeptide. cDNA sequence analysis and Southern blot analysis of pea nuclear DNA identifies at least three genes encoding GS in pea which are related but distinct in structure and in vivo pattern of expression.

show abstract

Factors affecting the separation of photosynthetically competent chloroplasts in gradients of silica sols

Cited by 79 publications

References 21 publications

Use of Silica Sol Step Gradients to Prepare Bundle Sheath and Mesophyll Chloroplasts from Panicum maximum

Use of Silica Sol Step Gradients to Prepare Bundle Sheath and Mesophyll Chloroplasts from Panicum maximum

Senescence-Induced, Thylakoid-Bound Diisopropylfluorophosphate-Binding Protein in Spinach

Glutamine synthetase genes of pea encode distinct polypeptides which are differentially expressed in leaves, roots and nodules.

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