Isolation and characterization of peanut spherosomes

Spherosomes of cotyledons of germinating peanuts (Arachis hypogea L.) were examined by electron microscopy and found to be particles about 1.0 to 2.0 μ in diameter bounded by a limiting membrane. Isolated spherosomes appear similar to spherosomes in situ. The isolated spherosomes are composed of 98.1% total lipids, 0.77% phospholipid and 1.27% protein by dry weight. The amounts of protein and phospholipid associated with the isolated spherosomes are sufficient to account for limiting membranes. Spherosomes amply account for the lipid in a peanut cotyledon. The activity of lipase and fatty acyl-Coenzyme A synthetase is not associated with the isolated spherosomes. This suggests that peanut spherosomes are principal sites of lipid storage but not of lipid degradation.     

Light and electron microscopy of virus inclusions inAmaranthus lividus cells

Summary Amaranthus plants infected with a virus of rod-shaped particles showed under the light microscope intracytoplasmic amorphous and crystalline inclusions.The submicroscopic organization of mesophyll cells from infectedAmaranthus leaves by electron microscopy is described. Besides big crystalline inclusions, long dark inclusions correspondent to needle-like inclusions observed by light microscopy are definable in the cytoplasm. The amorphous inclusion bodies were formed by an overgrown protrusion of vacuolate cytoplasm containing virus particles, long very dark stained inclusions forming dense bands and rings, normal elements of the cytoplasm such as mitochondria, endoplasmic reticulum and ribosomes, and some spherosomes. Inclusions and virus particles were not found in chloroplasts, mitochondria or nuclei of infected cells.     

THE SPHEROSOMES AND THE RESERVE FAT IN PLANT CELLS

The deposition of reserve fat is studied in plants with a high, medium, or low lipid content, and is contrasted with the spherosomes of the same cells. In storage tissue with a high lipid content the reserve triglycerides have the form of comparatively large globules which are quite distinct from the spherosomes. In plants with a medium lipid content the reserve fat appears in the form of granules, globules, or oil-plasm, and as a homogeneous, interstitial deposition between the protein bodies. Plants with a low lipid content contain a very large number of spherosomes and only very few small sudanophil globules. The spherosomes and the reserve fat represent distinctly separate entities.     

DIFFERENTIATION OF INTRACAPSULAR CELLS IN THE SPOROPHYTE OF SPHAEROCARPOS DONNELLII

Ultrastructural and histochemical changes during intracapsular cell differentiation in the premeiotic sporophyte of the liverwort Sphaerocarpos donnellii Austin were studied. From an initially undifferentiated meristematic tissue, spore mother cells and nutritive cells become differentiated. The first indications of ultrastructural differentiation into two cell types are the accumulation of lipid within spherosomes and the occurrence of plastid tubules in the presumptive spore mother cells. Once differentiated the two cell types are clearly distinguishable on the basis of cytoplasmic vacuolation, stored food reserve, and cell and nuclear size. The mature spore mother cell contains many spherosomes, small vacuoles, starch-containing plastids, and a large central nucleus. The mature nutritive cell, on the other hand, is extremely vacuolate and contains large, starch-filled plastids, a few spherosomes, and a small nucleus. A previously undescribed type of cell was observed in developing sporophyte capsules. This cell is located peripherally in the capsule and degenerates during differentiation of spore mother cells and nutritive cells.     

Ultrastructural Details in Germinating Sporangiospores of Rhizopus stolonifer and Rhizopus arrhizus

Electron microscope examination of sporangiospore sections from Rhizopus stolonifer (Ehrenb. ex Fr.) Lind. and R. arrhizus Fischer revealed details on intracellular organization not previously reported. Aldehyde fixation followed by chromeosmium postfixation permitted clear depiction of ribosomes hitherto unrevealed in these cells. Mitochondria were diversiform. Spore wall structures in the two species were generally similar, but outer contours differed sufficiently to permit easy species identification in examination of sections. The spores of both species abounded in cytosomes, corresponding in size, shape, and heavy-metal "stain" affinities to spherosomes in cells of higher plants. The osmiophilic response of these spherosome-like inclusions was intensified by treatment of sections with thiocarbohydrazide solution and subsequent application of aqueous osmium tetroxide, which strengthens an assumption that they are lipid-rich. The margins of the spherosome-like inclusions in lead citrate-stained sections included dense particles, about 60 A across, whose crystalline-like arrangements suggested that protein as well as lipid was present. Frequent and close associations between the spherosome-like inclusions and various cell membranes suggested that such bodies participate in membrane elaboration during germination.     

SPHEROSOMES AND MITOCHONDRIA IN THE LIVING FUNGAL CELL

Study of living hyphae of Fusarium oxysporum Schlect., Fomes annosus (Fries) Cooke, Ceratocystis fagacearum (Bretz) Hunt, Basidiobolus ranarum Eidam, and Mycotypha microspora Fenner with phase contrast revealed that these fungi have spherosomes similar to those in vascular plants. The spherosomes are conspicuous in the hyphal tip, suggesting some function other than fat synthesis. It may be that the Woronin bodies reported by other workers are spherosomes. Mitochondria in these fungi are highly pleomorphic and exhibit saltatory movement. They often interact with nuclei in a manner suggesting close membrane contact.     

Membranous appendices of spherosomes (oleosomes)

Spherosomes (oleosomes) of cotyledons of rape (Brassica napus L.), sunflower (Helianthus annuus L.), and watermelon (Citrullus vulgaris, Schrad.) seedlings are delimited by a half unit membrane that appears to be continuous with each of the osmiophilic layers of a tripartite unit membrane forming a handlelike appendix of the spherosomes. Prior to any noticeable utilization of the spherosomal storage fat, ribosomes were found to be attached to these handles. At later stages appendices of the spherosomes are smooth, showing a diameter of about 22 nm that greatly exceeds the thickness of any other unit membrane profiles identical in structure and diameter osomes appears to be continuous with the thick lipid layer of the handles. In intermediate stages of fat depletion the spherosomal bodies become invaginated with cytoplasmic material. Finally vesicles with cytoplasmic contents surrounded by a membrane with a typically thick lipid layer are left in the cells. Membrane profiles indentical in structure and diameter to the spherosomal appendices were also present in electron micrographs of the lipolytic membrane fraction recovered from sucrose density gradients after centrifugation of a microsomal cell fraction. The ultrastructural observations are taken for evidence that the spherosomal appendices represent the lipase-carrying membranes isolated previously (Theimer and Rosnitschek, 1978). A novel hypothesis for development and utilization of fat-storing spherosomes is also proposed.     

MEMBRANOUS STRUCTURES IN YEASTS

1. Most yeast cells carrying out active respiration have spherical or ellipsoidal mitochondria, with plate-like cristae. 2. Cytoplasmic petite strains of Saccharomyces cerevisiae have aberrant mitochondria, often containing whorled membranes. Mutants with deficiencies in the tricarboxylic acid cycle have mitochondria which appear normal when the cells are grown in low levels of glucose. 3. Cells of normal and petite S. cerevisiae grown strictly anaerobically show no recognizable mitochondrial profiles. 4. Carbon substrates which can only be respired promote the development of well-defined mitochondria. In certain facultatively anaerobic yeasts respiration is suppressed by glucose and the mitochondria under these conditions are large, pleomorphic and few in number. Other fermentable carbohydrates do not give this repression. 5. A number of antibacterial antibiotics, which inhibit mitochondrial protein synthesis, cause a disorganization of the mitochondrial cristae. 6. In yeast cells adapting from anaerobic to aerobic conditions mitochondria appear to develop from proliferations of the endoplasmic reticulum, which become progressively more organized. 7. Vacuoles often contain granular material, but in S. cerevisiae the vacuole, which has been described as a lysosome, frequently contains myelin-like lipid inclusions. The material in these inclusions is apparently derived from spherosomes. 8. Endoplasmic reticulum, orientated parallel to the plasmalemma, may be associated with fermentative ability in certain facultatively anaerobic yeasts. Endoplasmic reticulum is also actively involved in the budding process. 9. Normally the yeast-cell plasmalemma shows only minor convolutions, but in chloramphenicol-grown Rhodotorula glutinis the plasmalemma produces vesicular structures termed ‘paramural bodies’. 10. The yeast nuclear membrane has about 200 pores occupying 6–8 % of the total surface area. The nuclear membrane remains intact during mitotic division in yeasts until the daughter nuclei separate.     

STIGMA,STYLE, AND OBTURATOR OF ORNITHOGALUM CAUDATUM (LILIACEAE) AND THEIR FUNCTION IN THE REPRODUCTIVE PROCESS

Transmitting tissue in Ornithogalum is divided into three regions corresponding to classical divisions of the gynoecium: stigma, style, and ovary. The stigma differentiates from epidermal cells of the stylar apex. These cells form the stigmal papillae and have dense cytoplasm with abundant ER and lipid bodies. Papillae have walls with small transfer-ingrowths. At floral receptivity, papillae secrete a small amount of surface exudate. Epidermal cells of the style contain numerous spherosomes and have thin filaments of cytoplasm traversing the central vacuole. The stylar cortex is composed of 3-6 layers of parenchyma cells which contain numerous spherosomes and often have secondary vacuoles. Vascular tissue in the style consists of one collateral bundle in each lobe. Cells of the epidermal layer lining the stylar canal are secretory. They are initially vacuolate but fill progressively with dense cytoplasm as their secretory activity increases. Secretory activity occurs in three phases, each characterized by a particular organelle population and secretory product. At anthesis, the canal is filled with an exudate consisting of carbohydrate, protein, and lipid. In the ovary, the obturator differentiates from cells at the base of the funiculus and the tip of the carpel margins. It forms a pad of tissue which covers most of the former placenta. The obturator is secretory and produces a surface exudate. We believe our observations on Ornithogalum support the hypothesis that all transmitting tissue is of the same morphological origin and that it provides nutritive and chemotropic factors for pollen tube growth.     

Trypanosoma cruzi epimastigotes are able to store and mobilize high amounts of cholesterol in reservosome lipid inclusions

Background

Reservosomes are lysosome-related organelles found in Trypanosoma cruzi epimastigotes. They represent the last step in epimastigote endocytic route, accumulating a set of proteins and enzymes related to protein digestion and lipid metabolism. The reservosome matrix contains planar membranes, vesicles and lipid inclusions. Some of the latter may assume rectangular or sword-shaped crystalloid forms surrounded by a phospholipid monolayer, resembling the cholesterol crystals in foam cells.

Methodology/Principal Findings

Using Nile Red fluorimetry and fluorescence microscopy, as well as electron microscopy, we have established a direct correlation between serum concentration in culture medium and the presence of crystalloid lipid inclusions. Starting from a reservosome purified fraction, we have developed a fractionation protocol to isolate lipid inclusions. Gas-chromatography mass-spectrometry (GC-MS) analysis revealed that lipid inclusions are composed mainly by cholesterol and cholesterol esters. Moreover, when the parasites with crystalloid lipid-loaded reservosomes were maintained in serum free medium for 48 hours the inclusions disappeared almost completely, including the sword shaped ones.

Conclusions/Significance

Taken together, our results suggest that epimastigote forms of T. cruzi store high amounts of neutral lipids from extracellular medium, mostly cholesterol or cholesterol esters inside reservosomes. Interestingly, the parasites are able to disassemble the reservosome cholesterol crystalloid inclusions when submitted to serum starvation.     

Development of aleurone and sub-aleurone layers in maize

Electron-microscope studies indicate that the aleurone tissue of maize (Zea mays L.) starts developing approximately 10–15 days after pollination in stocks that take ca. 40 days for the aleurone to mature completely. Development commences when specialized endosperm cells adjacent to the maternal nucellar layer start to differentiate. Differentiation is characterized by the formation of aleurone protein bodies and spherosomes. The protein bodies of the aleurone layer have a vacuolar origin whereas the protein bodies of the immediate underlying endosperm cells appear to develop from protrusions of the rough endoplasmic reticulum. Thus, two morphologically and developmentally distinct types of protein bodies are present in these adjacent tissues. The spherosomes of the aleurone layer form early in the development of this tissue and increase in number as the tissue matures. During the final stages of maturation, these spherosomes become closely apposed to the aleurone grains and the plasma membrane. No further changes are apparent in the structure of the aleurone cells after 40 days from pollination when the caryopsis begins to desiccate.     

PHYSIOLOGICAL IMPLICATIONS OF VICIA FABA AND NICOTIANA TABACUM GUARD-CELL ULTRASTRUCTURE

The guard cells of Vicia faba and Nicotiana tabacum contain numerous mitochondria, elements of endoplasmic reticulum, spherosomes, and peroxisome-like microbodies. A full ribosomal complement appears in young but not in fully mature guard cells. Numerous small lipid droplets external to the plasmalemma were noted in mature Vicia guard cells. Chloroplasts were found in both epidermal and guard cells of both species. Full photosynthetic capacity was indicated by the grana fretwork of guard-cell chloroplasts. A specialized peripheral reticulum was observed in the guard-cell chloroplasts of Vicia. Plasmodesmata were observed in both walls between sister guard cells and between guard and epidermal cells. In the latter case plasmodesmata were found primarily in pit fields of transverse walls. It is postulated that the small volume of guard cells allows them an osmotic advantage over larger neighboring cells in generating turgor.     

Distribution of lipids from the yolk to the tissues during development of the water python (<Emphasis Type="Italic">Liasis fuscus</Emphasis>)

Energy metabolism during embryonic development of snakes differs in several respects from the patterns displayed by other reptiles. There are, however, no previous reports describing the main energy source for development, the yolk lipids, in snake eggs. There is also no information on the distribution of yolk fatty acids to the tissues during snake development. In eggs of the water python (Liasis fuscus), we report that triacylglycerol, phospholipid, cholesteryl ester and free cholesterol, respectively, form 70.3%, 14.1%, 5.7% and 2.1% of the total lipid. The main polyunsaturate of the yolk lipid classes is 18:2n-6. The yolk phospholipid contains 20:4n-6 and 22:6n-3 at 13.0% and 3.6% (w/w), respectively. Approximately 10% and 30% of the initial egg lipids are respectively recovered in the residual yolk and the fat body of the hatchling. A major function of yolk lipid is, therefore, to provision the neonate with large energy reserves. The proportion of 22:6n-3 in brain phospholipid of the hatchling is 11.1% (w/w): this represents only 0.24% of the amount of 22:6n-3 originally present in the egg. This also contrasts with values for free-living avian species where the proportion of DHA in neonatal brain phospholipid is 16–19%. In the liver of the newly hatched python, triacylglycerol, phospholipid and cholesteryl ester, respectively, form 68.2%, 7.7% and 14.3% of total lipid. This contrasts with embryos of birds where cholesteryl ester forms up to 80% of total liver lipid and suggests that the mechanism of lipid transfer in the water python embryo differs in some respects from the avian situation.Abbreviations ARA arachidonic acid - DHA docosahexaenoic acidCommunicated by G. Heldmaier     

Viral DNA Synthesis In Vitro with the Inclusions Isolated from Adenovirus 12-Infected Cells

A fraction defined as the inclusions was isolated by banding in CsCl gradients from nuclei of adenovirus 12-infected KB cells. When examined by electron microscopy, the isolated inclusions were relatively homogeneous, finely granular materials of moderate electron density, possibly representing the disintegrated type II or IV inclusions. The conditions of endogenous DNA synthesis in vitro with the inclusions were determined. The product of DNA synthesis in vitro with the inclusions was mainly viral and scarcely cellular, as revealed by DNA-DNA hybridization and methylated albumin kieselgur column chromatography. However, viral DNA synthesized in vitro was smaller (18 S, 22 S) than viral DNA in virions (31 S, 34 S) in neutral and alkaline sucrose gradients. Effects of various treatment of the inclusions on the DNA-synthesizing activity showed that phospholipase C inhibited the activity efficiently. The in vitro DNA synthesis was stimulated by addition of the cytoplasmic extract from adenovirus 12-infected cells and not that from unifected cells. The analysis of the composition of the inclusions showed that the inclusions contained DNA, protein, phospholipid and a small amount of RNA and carbohydrate.     

Effect of age on the membrane lipid composition of Streptococcus sanguis

The cell membrane of Streptococcus sanguis contains three classes of lipid: neutral lipid, glycolipid and phospholipid. A striking difference in membrane lipid composition between cells in the exponential and in the stationary phases of growth was observed. During the exponential phase, approx. 37–45%, 14–19% and 37–45% of the lipids synthesized were found to be neutral lipid, glycolipid and phospholipid, respectively. The amount of lipid synthesized reached a maximum at the early stationary phase. The amount of phospholipid drastically declined thereafter and that of neutral lipid slightly declined. In contrast, the amount of glycolipid markedly increased and exceeded the amount of phospholipid. The phospholipid present during the exponential phase was found to be mainly phosphatidylglycerol (82–88%) and a small amount of cardiolipin (12–18%). At the stationary phase, the amount of phosphatidylglycerol greatly decreased and reached approx. 16% of that in the early stationary phase, while cardiolipin steadily increased and became the major phospholipid in the late stationary phase. The glycolipid was found to be composed of mainly mono- and diglucosyldiglycerides. At the end of the experiment (after 8 h incubation), the distribution of lipids was found to be: neutral lipid, 46%; glycolipid (monoglucosyldiglyceride, 28%; diglucosyldiglyceride, 13%) 41%; and phospholipid (phosphatidylglycerol, 3%, cardiolipin, 8%) 13%.     

The ontogeny of lipid bodies (spherosomes) in plant cells

Maturing embryos of 16 oil plants, anise suspension culture cells, and Neurospora crassa cells were prepared for electron microscopy at different stages during massive lipid accumulation. Lipid-rich structures of certain species were best preserved by dehydration of fixed tissues in ethanol without propylene oxide, embedding in Spurr's Medium, and polymerization at room temperature. In all cells examined, spherical lipid bodies (spherosomes) showed a moderately osmiophilic, amorphous matrix and displayed a delimiting half-unit membrane when sectioned medially. Associations with the endoplasmic reticulum (ER) were viewed at any stage during lipid body development but with different frequency in the different plant species. Plastids of fat-storing cells exhibited conspicuously undulate outer and inner envelope membranes that formed multiple contact sites with each other and protuberances into both cytoplasm and stroma. Some species, e.g., Linum, have plastids with tubular structures that connect the inner membrane to the thylakoid system; in addition, in the stroma vesicles fusing with or apparently passing through the envelope were observed. The outer envelope membrane may be associated with ER-like cytoplasmic membrane structures. In addition, lipid bodies of various sizes were found in contact with the plastid envelope. The ultrastructural observations are interpreted to match the published biochemical evidence, indicating that both plastids and ER may be involved in the synthesis of storage lipids and lipid body production.     

Involvement of glyoxysomal lipase in the hydrolysis of storage triacylglycerols in the cotyledons of soybean seedlings

The total cotyledon extract of soybean (Glycine max [L.] Merr. var. Coker 136) seedlings underwent lipolysis as measured by the release of fatty acids. The highest lipolytic activity occurred at pH 9. This lipolytic activity was absent in the dry seeds and increased after germination concomitant with the decrease in total lipids. Using spherosomes (lipid bodies) isolated from the cotyledons during the peak stage of lipolysis (5-7 days) as substrates, about 40% of the lipase activity was found in the glyoxysomes after organelle breakage had been accounted for; the remaining activity was distributed among other subcellular fractions but none was found in the spherosomal fraction. The glyoxysomal lipase had maximal activity at pH 9, and catalyzed the hydrolysis of tri-, di-, and monoacylglycerols of linoleic acid, the most abundant fatty acid in soybean. The spherosomes contained a neutral lipase that could hydrolyze monolinolein and N-methylindoxylmyristate, but not trilinolein. This spherosomal lipase activity dropped off rapidly during early seedling growth, preceding lipolysis. Spherosomes isolated from either dry or germinated seeds did not possess lipolytic activity, and spherosomes from germinated seeds but not from dry seeds could serve as substrates for the glyoxysomal lipase. It is concluded that the glyoxysomal lipase is the enzyme catalyzing the initial hydrolysis of storage triacylglycerols.     

INCLUSIONS OF THE PROPLASTIDS AND VACUOLES IN THE SHOOT APICES OF BRYOPHYLLUM AND KALANCHOË

Spherical, membrane-bound inclusions occur in the proplastids and vacuoles of cells of Bryophyllum and Kalanchoë shoot apices. Evidence is presented indicating that the inclusions arise by the accumulation of material within the cisternae of certain tubular proplastid membranes and are then transferred to vacuoles. The results obtained from electron microscopy and from histochemical studies indicate that the contents of the inclusions are predominantly lipid.     

Lipid synthesis during reinitiation of growth from stationary phase cultures of Candida albicans

Lipid synthesis has been studied in the dimorphic fungus Candida albicans. ¹⁴C-acetate incorporation into lipid material was used to measure new lipid synthesis in two cultures in which either yeast or mycelial growth was initiated from stationary phase yeast cells. When resuspended in fresh medium at 37 °C, cells resume growth and change morphology while at 30 °C cells resume budding growth. When resuspended at the appropriate temperature, both yeast and germ tube cultures immediately incorporated ¹⁴C-acetate into lipid material. The labeled lipid was more or less evenly divided between neutral and phospholipid. Phosphatidyl choline was the major phospholipid fraction and along with phosphatidyl ethanolamine accounted for 60–65 % of the total phospholipid. Lipid synthesis during growth initiation of either morphology showed a similar pattern, with no significant differences observed in neutral or phospholipid or phospholipid components between yeast and mycelial forms.     

Mitochondrial polymorphism. IV. Phospholipid composition of mitochondria of a wheat hybrid and its parents

Mitochondria of young seedlings of wheat genotypes 28, 31MS, and 31MS |MX 28 differed in total lipid and phospholipid. Hybrid mitochondria had more lipid and phospholipid than did the parents, and the three genotypes differed in fatty acid composition of the phospholipid fraction. Hybrid mitochondria exhibited heterosis in cytochrome oxidase activity. Although depletion of phospholipid greatly reduced cytochrome oxidase activity in hybrid mitochondria, the heterotic property was preserved. Regulatory aspects of lipid and phospholipid in mitochondria of the hybrid and complementing parental mixture are considered.This research was supported by DeKalb Ag-Research Inc. and in part by grant A-338 from Robert A. Welch Foundation of Houston, Texas.The preceding paper in this series is Sarkissian, I. V., and Srivastava, H. K. (1971). Biochem. Genet. 5:57.