[16] Isolation of plastids in density gradients of percoll and other silica sols

Price, Colin; Cushman, John C.; Mendiola-Morgenthaler, L.; Reardon, Ellen M.

doi:10.1016/0076-6879(87)48018-x

Cited by 23 publications

(15 citation statements)

References 32 publications

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“…Similar results were reported by numerous other investigators for various plastid preparations (16,18,19 In view of their high rates of ALA to Proto conversion, their low level of nonplastidic subcellular organelle contamination, including mitochondrial contamination, and the very low level of ALA to Proto conversion exhibited by mitochondria (Table I), Percoll-purified etiochloroplasts were deemed acceptable as a starting point for further investigations of the intraplastidic localization of the ALA to Proto biosynthetic activity.…”

Section: Evaluation Of the Purity Of Percoll-purified Plastidssupporting

confidence: 61%

Intraplastidic Localization of the Enzymes That Convert δ-Aminolevulinic Acid to Protoporphyrin IX in Etiolated Cucumber Cotyledons

Lee¹,

Ball²,

Rebeiz³

1991

Plant Physiol.

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The intraplastidic localization of the enzymes that catalyze the conversion of 6-aminolevulinic acid (ALA) to protoporphyrin IX (Proto) is a controversial issue. While some researchers assign a stromal location for these enzymes, others favor a membranebound one. Etiochloroplasts were isolated from etiolated cucumber cotyledons (Cucumis sativus, L.) by differential centrifugation and were purified further by Percoll density gradient centrifugation. Purified plastids were highly intact, and contamination by other subcellular organelles was reduced five-to ninefold in comparison to crude plastid preparations. Most of the ALA to Proto conversion activity was found in the plastids. On a unit protein basis, the ALA to Proto conversion activity of isolated mitochondria was about 2% that of the purified plastids, and could be accounted for by contamination of the mitochondrial preparation by plastids. Lysis of the purified plastids by osmotic shock followed by high speed centrifugation, yielded two subplastidic fractions: a soluble clear stromal fraction and a pelleted yellowish one. The stromal fraction contained about 11% of the plastidic ALA to Proto conversion activity while the membrane fraction contained the remaining 89%. The stromal ALA to Proto conversion activity was in the range of stroma contamination by subplastidic membrane material. Complete solubilization of the ALA to Proto activity was achieved by high speed shearing and cavitation, in the absence of detergents. Solubilization of the ALA to Proto conversion activity was accompanied by release of about 30% of the membrane-bound protochlorophyllide. It is proposed that the enzymes that convert ALA to Proto are loosely associated with the plastid membranes and may be solubilized without the use of detergents. It is not clear at this stage whether the enzymes are associated with the outer or inner plastid membranes and whether they form a multienzyme complex or not.It is presently acknowledged that the enzymes that convert ALA3 to protochlorophyll(ides) and Chl are located in the plastids (5,6,20,22,23). The intraplastidic localization of

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Section: Evaluation Of the Purity Of Percoll-purified Plastidssupporting

confidence: 61%

Intraplastidic Localization of the Enzymes That Convert δ-Aminolevulinic Acid to Protoporphyrin IX in Etiolated Cucumber Cotyledons

Lee¹,

Ball²,

Rebeiz³

1991

Plant Physiol.

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“…Intact chloroplasts were isolated by Percoll gradient centrifugation (Price et al, 1987) and lysed by osmotic shock in 5 mM Tricine-KOH and 5 mM MgCl 2 , pH 7.9. The lysate was fractionated into stroma and thylakoid fractions by centrifugation at 3000g for 5 min.…”

Section: Chloroplast Isolation and Fractionationmentioning

confidence: 99%

Reduced Levels of Chloroplast FtsH Protein in Tobacco Mosaic Virus–Infected Tobacco Leaves Accelerate the Hypersensitive Reaction

et al. 2000

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In tobacco cultivars resistant to tobacco mosaic virus (TMV), infection results in the death of the infected cells accompanying the formation of necrotic lesions. To identify the genes involved in this hypersensitive reaction, we isolated the cDNA of tobacco DS9, the transcript of which decreases before the appearance of necrotic lesions. The DS9 gene encodes a chloroplastic homolog of bacterial FtsH protein, which serves to maintain quality control of some cytoplasmic and membrane proteins. A large quantity of DS9 protein was found in healthy leaves, whereas the quantity of DS9 protein in infected leaves decreased before the lesions appeared. In transgenic tobacco plants containing less and more DS9 protein than wild-type plants, the necrotic lesions induced by TMV were smaller and larger, respectively, than those on wild-type plants. These results suggest that a decrease in the level of DS9 protein in TMV-infected cells, resulting in a subsequent loss of function of the chloroplasts, accelerates the hypersensitive reaction. INTRODUCTIONThe hypersensitive reaction (HR) is a plant defense system against pathogen attack. HR is defined as the rapid death of infected cells accompanying formation of necrotic lesions in which pathogens are thought to be enclosed (Goodman and Novacky, 1994). The HR of tobacco to tobacco mosaic virus (TMV) occurs in cultivars carrying the resistance gene N ( NN tobacco) but not in cultivars lacking the N gene ( nn tobacco) (Holmes, 1938; Whitham et al., 1994). Induction of the N gene-mediated HR requires the temperature to be below 22 to 26 Њ C (Kassanis, 1952;Shimomura, 1971; Weststeijn, 1981), suggesting that the gene can function at temperatures below this range.We previously observed that when TMV-inoculated leaves were treated with heat shock or actinomycin D, necrotic lesions were induced in not only NN but also in nn tobacco, even at 30 Њ C, a nonpermissive temperature for the N gene (Shimomura and Ohashi, 1971;Ohashi and Shimomura, 1972). In the actinomycin D-treated leaves in which necrotic lesions were induced, the synthesis of nucleic acid in the host plant was greatly suppressed, whereas the synthesis of TMV RNA was suppressed only slightly (Shimomura and Ohashi, 1980). In contrast to treatment with actinomycin D, treatment with other chemicals, including rifampicin, 2-thiouracil, and cycloheximide, which suppress RNA or protein synthesis in both the TMV and the host plant, did not induce necrotic lesions to form (Ohashi and Shimomura, 1972). Given that actinomycin D forms intercalation complexes with DNA, thereby blocking transcription (Waring, 1981), and that heat shock suppresses transcription of housekeeping genes (Lindquist, 1986;Nover et al., 1989), we hypothesized that suppressing the transcription of certain housekeeping genes in NN tobacco plants after infection with TMV could induce the HR.To isolate such candidate genes, we used a synchronous HR-inducing system based on a temperature shift. Incubation at 30 Њ C of TMV-infected NN tobacco leaves results in 1 Curr...

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“…Chloroplasts were isolated from 14-day-old seedlings of fava bean plants according to the procedure described by Price et al (1987). They were lysed by sonication in the extraction buffer (50 mM Tris, pH 7.5, 4 mM EDTA, 5 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 5 ng/mL leupeptin).…”

Section: Purification Of the Chloroplast-localized Cyclophilinmentioning

confidence: 99%

pCyP B: a chloroplast-localized, heat shock-responsive cyclophilin from fava bean.

1994

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When the immunosuppressants cyclosporin A (CsA) and FK506 bind to their intracellular receptors (immunophilins), they form complexes that bind to calcineurin and block calcineurin-dependent signaling pathways in immune cells. Previously, we reported that higher plants also express immunophilins and have a Ca2+-dependent signaling pathway sensitive to immunophilin-ligand complexes. Based on an N-terminal peptide sequence of a chloroplast-localized cyclophilin (pCyP B), we isolated a cDNA clone encoding the preprotein of the cyclophilin. The deduced amino acid sequence of this cDNA starts with a putative transit sequence for chloroplast targeting. The mature pCyP B protein has rotamase activity with low-substrate specificity. Enzyme activity was inhibited by CsA with an inhibition constant of 3.9 nM. Similar to otherCyPs from mammalian cells, pCyP B, when complexed with CsA, inhibited the phosphatase activity of bovine calcineurin. The mRNA leve1 of pCyP B was high in leaf tissues but was not detectable in roots. Expression of the transcript in the leaf tissues was regulated by light and induced by heat shock. These findings illustrate the conserved nature of cyclophilin proteins among all of the eukaryotes and suggest that cyclophilins have a unique mode of regulation in higher plants.

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[16] Isolation of plastids in density gradients of percoll and other silica sols

Cited by 23 publications

References 32 publications

Intraplastidic Localization of the Enzymes That Convert δ-Aminolevulinic Acid to Protoporphyrin IX in Etiolated Cucumber Cotyledons

Intraplastidic Localization of the Enzymes That Convert δ-Aminolevulinic Acid to Protoporphyrin IX in Etiolated Cucumber Cotyledons

Reduced Levels of Chloroplast FtsH Protein in Tobacco Mosaic Virus–Infected Tobacco Leaves Accelerate the Hypersensitive Reaction

pCyP B: a chloroplast-localized, heat shock-responsive cyclophilin from fava bean.

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