Isolation of Plastids from Sunflower Cotyledons during Germination

Plastids from cotyledons of sunflower (Helianthus annus L.) seedlings, germinated in the dark or in the light, were isolated by isopycnic sucrose density gradient centrifugation. At all stages of development the whole plastids contained triose phosphate isomerase, NADPH-glyoxylate reductase, and l-dihydroxyphenylalanine oxidase, which were used as marker enzymes. At the beginning of germination the isopycnic density of whole plastids (proplastids) was about 1.22 g cm⁻³. During development of proplastids into etioplasts in the dark, their isopycnic density increased to 1.26 g cm⁻³. During exposure of germinating seedlings to white light for 2 days, the isopycnic density of whole plastids decreased from 1.26 to 1.22 g cm⁻³. These changes in isopycnic density of plastids on sucrose density gradients are consistent with changes in the plastid ultrastructure caused by the protein-rich prolamellar body or by the lipid-rich thylakoids. Broken plastids (thylakoids), determined by the main peak of chlorophyll, increased in isopycnic density from less than 1.14 to about 1.17 g cm⁻³ during illumination. During germination no major changes occurred in the isopycnic density of mitochondria. Microbodies had an isopycnic density of 1.24 g cm⁻³ in very early stages of germination, and their density increased to 1.265 g cm⁻³, when glyoxysomal enzymes reached maximum development.     

Polyribosomes from Peas: V. An Attempt to Characterize the Total Free and Membrane-bound Polysomal Population

Attempts were made to isolate and characterize the total population of free and membrane-bound polysomes from the elongating region of dark-grown pea stems (Pisum sativum L.). Partial separation of free from membrane-bound polysomes was achieved by relatively low speed centrifugation of the homogenate. Complete separation was not achieved. Based on analysis of the rRNA content of various subcellular fractions, fractionated tissue yielded greater than 95% of the rRNA found in whole tissue. Approximately 45% of the ribosomal material was membrane-bound (released by detergent) and was found in the “wall” (13%), the “nuclear” pellet (2%), and the “mitochondrial” pellet (29%). The remaining 55%, consisting primarily of free polysomes, could be recovered free from membranous material by sedimentation through a dense (700 mg/ml) sucrose pad for 90 hours. The advantages and disadvantages of using sucrose pads for the separation of free and membrane-bound polysomes are discussed.     

Boophilus microplus: characterization of larval phosphomonoesterases and isolation of subcellular fractions with high phosphatase activity.

Phosphomonoesterase activity was determined for a 115,000g pellet and soluble fractions resulting from a subcellular fractioning of a homogenate of larval Boophilus microplus. Both fractions showed maximum phosphatase activity at pH 5.5 and 10. Acid phosphatase (EC 3.1.3.2) activity was found to be greatest in the soluble fraction. When the reaction rate was plotted against homogenate concentration, the soluble acid phosphatase deviated from the linear relationship. For both fractions different thermostability patterns were obtained, inactlvation beginning for the alkaline phosphatase (EC 3.1.3.1) at 45–55 C. When the effect of substrate concentration on activity was studied, deviations from the typical hyperbolic behavior were observed. Homogenization of larvae with 5 mm EDTA buffer failed to yield a low-speed pellet with high alkaline phosphatase activity, as it is expected if absorptive structures sediment. Moreover, total alkaline phosphatase activity recovered by this method is significantly lower than activity recovered when homogenization is carried out without EDTA. Alternately, homogenization with 10 mM Tris buffer and 0.25 M sucrose gave 27,000g and 115,000g fractions with high phosphatase activity when fractioned by centrifugation. Alkaline treatment of the 115,000g fraction with 10 mM Tris buffer, pH 7.8, failed to separate endoplasmic reticulum contaminants without loss of phosphatase activity. When the 115,000g fraction was centrifuged in a sucrose density gradient, two activity peaks, coincident for both acid and alkaline phosphatases, were obtained. Antigenic analysis showed the existence of similar antigenic determinants in both peaks “immunologically” presented in different ways.     

Isolation of furanoterpene-containing particles from Ceratocystis fimbriata-infected sweet potato root tissue

Furanoterpene-containing particles were isolated by centrifugation on a discontinuous Ficoll density gradient from a homogenate of the non-infected tissue adjacent to the infected region of Ceratocystis fimbriata-infected sweet potato root tissue. The particles were recovered at a relatively high ratio in the 2% Ficoll fraction, in which there was no contamination by mitochondria and only little by endoplasmic reticulum judging from the distribution of the activities of their marker enzymes and electron micrographs. Each particle was enveloped in a single membrane, 7-10 nm thick.     

Lipase Activities in Castor Bean Endosperm during Germination

Two lipases were found in extracts from castor bean (Ricinus communis L.) endosperm. One, with optimal activity at pH 5.0 (acid lipase), was present in dry seeds and displayed high activity during the first 2 days of germination. The second, with an alkaline pH optimum (alkaline lipase), was particularly active during days 3 to 5. When total homogenates of endosperm were fractionated into fat layer, supernatant, and particulate fractions, the acid lipase was recovered in the fat layer, and the alkaline lipase was located primarily in the particulate fraction. Sucrose density gradient centrifugation showed that the alkaline lipase was located mainly in glyoxysomes, with some 30% of the activity in the endoplasmic reticulum. When glyoxysomes were broken by osmotic shock and exposed to KCl, which solubilizes most of the enzymes, the alkaline lipase remained particulate and was recovered with the glyoxysomal “ghosts” at equilibrium density 1.21 g/cm³ on the sucrose gradient. Association of the lipase with the gly-oxysomal membrane was supported by the responses to detergents and to butanol. The alkaline lipase hydrolyzed only monosubstituted glycerols. The roles of the two lipases in lipid utilization during germination of castor bean are discussed.     

Highly purified plasma membranes from rat hepatocytes following rate-isopycnic zonal centrifugation

Plasma membranes from liver parenchymal cells were isolated by rate-isopycnic zonal centrifugation. A method is described for the Beckman size 15 zonal rotor. It involved preparation from a perfused liver of a parenchymal cell-enriched homogenate in isoosmotic sucrose. The nuclear fraction containing membranes was recovered by centrifugation. The resuspended pellet was applied on the gradient of the zonal rotor. The isolated membranes had the same isopycnic banding density as 37% sucrose (w/w). The specific activity of 5′-nucleotidase, a widely used plasma membrane marker, was 105 μmoles·(mg protein)^?1·h^?1 being enriched by a factor of 50 as compared with parenchymal cell homogenate. The plasma membrane fraction was free of the mitochondrial and lysosomal enzymes, succinate dehydrogenase and acid phosphatase. No DNA and 10 μg RNA per mg plasma membrane protein were found. The purity of the membranes and their morphological appearance were controlled by electron microscopy. The preparation consisting of large membrane sheets showed a considerable purification away from other cellular components. A comparison with similar methods indicates that plasma membranes of a higher degree of purity can be obtained from parenchymal cells.     

The isolation of endoplasmic reticulum from barley aleurone layers

Techniques for the isolation and purification of endoplasmic reticulum (ER) from aleurone layers of barley (Hordeum vulgare L.) were assessed. Neither differential centrifugation nor density gradient centrifugation of a homogenate separate the ER or other organelles of this tissue from the lipidcontaining spherosomes. Isopycnic sucrose gradient centrifugation of organelles first purified by molecular sieve chromatography on Sepharose 4B, however, results in separation of the organelles based on their differing buoyant densities. Manipulation of the magnesium concentration of the isolation media and density-gradient solutions affords isolation of ER at a density of 1.13–1.14 g cc^-1 and 1.17–1.18 g cc^-1. Electron microscopy shows that the membranes sedimenting at 1.13–1.14 g cc^-1 are devoid of ribosomes and are characteristic of smooth ER, while those sedimenting at 1.17–1.18 g cc^-1 are studded with ribosomes and have the features of rough ER. Endoplasmic reticulum isolated by isopycnic density gradient centrifugation can be further purified by rate-zonal centrifugation.Abbreviations EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - GA gibberellin - GA₃ gibberellic acid - Trizma tris(hydroxymethyl)aminomethane     

Cuscuta europaea plastid apparatus in various developmental stages: Localization of THF1 protein

It was generally accepted that Cuscuta europaea is mostly adapted to a parasitic lifestyle with no detectable levels of chlorophylls. We found out relatively high level of chlorophylls (Chls a+b) in young developmental stages of dodder. Significant lowering of Chls (a+b) content and increase of carotenoid concentration was typical only for ontogenetically more developed stages. Lower content of photosynthesis-related proteins involved in Chls biosynthesis and in photosystem formation as well as low photochemical activity of PSII indicate that photosynthesis is not the main activity of C. europaea plastids. Previously, it has been shown in other species that the Thylakoid Formation Protein 1 (THF1) is involved in thylakoid membrane differentiation, plant-fungal and plant-bacterial interactions and in sugar signaling with its preferential localization to plastids. Our immunofluorescence localization studies and analyses of haustorial plasma membrane fractions revealed that in addition to plastids, the THF1 protein localizes also to the plasma membrane and plasmodesmata in developing C. europaea haustorium, most abundantly in the digitate cells of the endophyte primordium. These results are supported by western blot analysis, documenting the highest levels of the THF1 protein in “get together” tissues of dodder and tobacco. Based on the fact that photosynthesis is not a typical process in the C. europaea haustorium and on the extra-plastidial localization pattern of the THF1, our data support rather other functions of this protein in the complex relationship between C. europaea and its host.     

Protein Synthesis in Cell-Free Extracts of Streptococcus faecalis

If a cell-free extract of Streptococcus faecalis is fractionated by way of a 10 to 70% sucrose gradient, at least three areas are found capable of protein synthesis having a density greater than can be accounted for by association of individual ribosomes. These areas represent distinct “polysome” peaks rather than random distribution of polymers of varied length. They appear to be membrane subunits. In addition there is a further particle with a density of about 150S, incapable of protein synthesis but which is capable of stimulating protein synthesis in some of the larger fractions found by gradient centrifugation.     

Isolation and characterization of plasma membrane from lactating bovine mammary gland

Plasma membranes were isolated from lactating bovine mammary gland. Two crude membrane fractions; medium/d 1.033 (light membrane) and 1.033/1.053 interfaces (heavy membrane), were obtained by Ficoll density gradient centrifugation of osmotically washed microsomal fraction. Two crude membranes were further purified separately by sucrose density gradient centrifugation. Both light and heavy membranes banded at a sucrose density of 1.14. The purified membranes appeared as heterogeneous smooth membrane vesicles on electron microscopy. The contaminating suborganelles were not detected. The yield of the purified membranes relative to the homogenate was 1.2%. The degree of purity of the membranes was shown by a great increase in the specific activity of 5′-nucleotidase over the homogenate of 20-fold for light membrane and of 16-fold for heavy membrane. The relative activities of Mg²⁺-ATPase, (Na⁺ + K⁺)-ATPase, γ-glutamyl transpeptidase, phosphodiesterase I, akaline phosphatase and xanthine oxidase were also high (12–18-times) and nearly 20% of these enzymes was recovered. The activity of marker enzyme for mitochondria, endoplasmic reticulum and Golgi apparatus was very low, while that of acid phosphatase for lysosome was relatively high (5-times). DNA and RNA contents were very low. The major polypeptides rich in other suborganelles were not detected profoundly in the membrane fraction and the polypeptide compositions in both light and heavy membranes were similar upon SDS-polyacrylamide gel electrophoresis.     

Isolation of amyloplasts from developing maize endosperm

Methods for the formation of protoplasts from developing maize endosperm and for the aqueous isolation of intact amyloplasts from such protoplasts are described. Protoplasts were obtained after incubating endosperm slices in a medium containing cellulase and pectolyase for 5 days at 4°C or 5 hours at 30°C. After purification in a Ficoll density gradient, the protoplasts were reptured by forcing the suspension through a Nitex mesh (20 micrometer) positioned at the lower end of a modified disposable syringe. The resulting filtrate was layered on a discontinuous Ficoll density gradient of 30, 15, and 10%. Each Ficoll solution contained 0.7 molar sucrose, 10 millimolar arginine, 10 millimolar dl-dithiothreitol, 50 millimolar 2-(N-morpholino)ethanesulfonic acid (pH 5.6), and 2 millimolar CaCl₂. After 3 hours in the cold, an amyloplast fraction 50 to 93% intact and free from cytoplasmic, mitochondrial, and glyoxysomal contamination was recovered in the 15% Ficoll layer. Amyloplast intactness was estimated by fluorescent microscopy and activity of certain amyloplast marker enzymes before and after rupture of the amyloplast membrane. Starch branching enzyme, ADPG-pyrophosphorylase, and nitrite reductase were used as amyloplast marker enzymes.     

Diversity and Distribution in Hypersaline Microbial Mats of Bacteria Related to Chloroflexus spp.

Filamentous bacteria containing bacteriochlorophylls c and a were enriched from hypersaline microbial mats. Based on phylogenetic analyses of 16S rRNA gene sequences, these organisms form a previously undescribed lineage distantly related to Chloroflexus spp. We developed and tested a set of PCR primers for the specific amplification of 16S rRNA genes from filamentous phototrophic bacteria within the kingdom of “green nonsulfur bacteria.” PCR products recovered from microbial mats in a saltern in Guerrero Negro, Mexico, were subjected to cloning or denaturing gradient gel electrophoresis and then sequenced. We found evidence of a high diversity of bacteria related to Chloroflexus which exhibit different distributions along a gradient of salinity from 5.5 to 16%.     

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.     

Polysaccharide-degrading Enzymes are Unable to Attack Plant Cell Walls without Prior Action by a "Wall-modifying Enzyme"

A study of the degradation of plant cell walls by the mixture of enzymes present in Pectinol R-10 is described. A “wall-modifying enzyme” has been purified from this mixture by a combination of diethylaminoethyl cellulose, Bio Gel P-100, and carboxymethyl cellulose chromatography. Treatment of cell walls with the “wall-modifying enzyme” is shown to be a necessary prerequisite to wall degradation catalyzed by a mixture of polysaccharide-degrading enzymes prepared from Pectinol R-10 or by an α-galactosidase secreted by the pathogenic fungus Colletotrichum lindemuthianum. The action of the “wall-modifying enzyme” on cell walls is shown to result in both a release of water-soluble, 70% ethanol-insoluble polymers and an alteration of the residual cell wall. A purified preparation of the “wall-modifying enzyme” is unable to degrade a wide variety of polysaccharide, glycoside, and peptide substrates. However, the purified preparation of wall-modifying enzyme has a limited ability to degrade polygalacturonic acid. The fact that polygalacturonic acid inhibits the ability of the “wall-modifying enzyme” to affect cell walls suggests that the “wall-modifying enzyme” may be responsible for the limited polygalacturonic acid-degrading activity present in the purified preparation. The importance of a wall-modifying enzyme in developmental processes and in pathogenesis is discussed.     

Preparation and characterization of envelope membranes from nongreen plastids

We have developed a reliable procedure for the purification of envelope membranes from cauliflower (Brassica oleracea L.) bud plastids and sycamore (Acer pseudoplatanus L.) cell amyloplasts. After disruption of purified intact plastids, separation of envelope membranes was achieved by centrifugation on a linear sucrose gradient. A membrane fraction, having a density of 1.122 grams per cubic centimeter and containing carotenoids, was identified as the plastid envelope by the presence of monogalactosyldiacylglycerol synthase. Using antibodies raised against spinach chloroplast envelope polypeptides E24 and E30, we have demonstrated that both the outer and the inner envelope membranes were present in this envelope fraction. The major polypeptide in the envelope fractions from sycamore and cauliflower plastids was identified immunologically as the phosphate translocator. In the envelope membranes from cauliflower and sycamore plastids, the major glycerolipids were monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and phosphatidylcholine. Purified envelope membranes from cauliflower bud plastids and sycamore amyloplasts also contained a galactolipid:galactolipid galactosyltransferase, enzymes for phosphatidic acid and diacylglycerol biosynthesis, acyl-coenzyme A thioesterase, and acyl-coenzyme A synthetase. These results demonstrate that envelope membranes from nongreen plastids present a high level of homology with chloroplasts envelope membranes.     

Genetic Distinctions among Clinical and Environmental Strains of Vibrio vulnificus

Vibrio vulnificus causes rare but frequently fatal septicemia associated with raw oyster consumption by persons with underlying hepatic or immune system dysfunction. The virulence potential of environmental reservoirs appears widely distributed, because most strains are virulent in animal models; however, several investigations recently demonstrated genetic divergence among strains from clinical versus environmental origin at independent genetic loci. The present study used PCR to screen DNA polymorphisms in strains from environmental (n = 35) or clinical (n = 33) sources, and genomic relationships were determined by repetitive extragenic palindromic DNA PCR (rep-PCR) typing. Significant (P < 0.01) association was observed for typical “clinical” or “environmental” polymorphism profiles based on strain origin. Most oyster isolates (88%), including all of those with the “environmental” profile, also formed a single rep-PCR genogroup. Clinical isolates within this group did not have the typical “clinical” profile. On the other hand, clinical isolates with the typical polymorphism profile were distributed among multiple rep-PCR genogroups, demonstrating greater genetic diversity than was evident by profiling genetic polymorphisms. Wound isolates were genetically distinct from typical blood isolates by all assays. Strains from an outbreak of wound infections in Israel (biotype 3) were closely related to several U.S. strains by rep-PCR, indicating potential reservoirs of emerging disease. Strains genetically related to blood isolates appeared to be relatively rare in oysters, as only one had the “clinical” polymorphism profile or clustered by rep-PCR. However, this study was not an extensive survey, and more sampling using rep-PCR for sensitive genetic discrimination is needed to determine the virulence potential of environmental reservoirs.     

Isolation of intact plastids from a range of plant tissues

A technique for the isolation of intact plastids from spinach (Spinacia oleracea) and pea (Pisum sativum) leaves, pea roots and castor bean (Ricinus communis) endosperm is described. This technique involves brief centrifugation of whole homogenates on density gradients. Intact plastids were located in the gradient by assaying for triose phosphate isomerase activity. Contamination of the plastic peak with mitochondria and microbodies was estimated by measurement of cytochrome oxidase and catalase, respectively. For three of the four tissues the level of contamination of the plastids by these organelles was 2% or less. The sedimentation behavior of microbodies from different tissues is discussed.     

Infection Density Dynamics of the Citrus Greening Bacterium “Candidatus Liberibacter asiaticus” in Field Populations of the Psyllid Diaphorina citri and Its Relevance to the Efficiency of Pathogen Transmission to Citrus Plants

Huanglongbing, or citrus greening, is a devastating disease of citrus plants recently spreading worldwide, which is caused by an uncultivable bacterial pathogen, “Candidatus Liberibacter asiaticus,” and vectored by a phloem-sucking insect, Diaphorina citri. We investigated the infection density dynamics of “Ca. Liberibacter asiaticus” in field populations of D. citri with experiments using field-collected insects to address how “Ca. Liberibacter asiaticus” infection density in the vector insect is relevant to pathogen transmission to citrus plants. Of 500 insects continuously collected from “Ca. Liberibacter asiaticus”-infected citrus trees with pathological symptoms in the spring and autumn of 2009, 497 (99.4%) were “Ca. Liberibacter asiaticus” positive. The infections were systemic across head-thorax and abdomen, ranging from 10³ to 10⁷ bacteria per insect. In spring, the infection densities were low in March, at ∼10³ bacteria per insect, increasing up to 10⁶ to 10⁷ bacteria per insect in April and May, and decreasing to 10⁵ to 10⁶ bacteria per insect in late May, whereas the infection densities were constantly ∼10⁶ to 10⁷ bacteria per insect in autumn. Statistical analysis suggested that several factors, such as insect sex, host trees, and collection dates, may be correlated with “Ca. Liberibacter asiaticus” infection densities in field D. citri populations. Inoculation experiments with citrus seedlings using field-collected “Ca. Liberibacter asiaticus”-infected insects suggested that (i) “Ca. Liberibacter asiaticus”-transmitting insects tend to exhibit higher infection densities than do nontransmitting insects, (ii) a threshold level (∼10⁶ bacteria per insect) of “Ca. Liberibacter asiaticus” density in D. citri is required for successful transmission to citrus plants, and (iii) D. citri attaining the threshold infection level transmits “Ca. Liberibacter asiaticus” to citrus plants in a stochastic manner. These findings provide valuable insights into understanding, predicting, and controlling this notorious citrus pathogen.