Targeting of oleosins to the oil bodies of oilseed rape (Brassica napus L.)

Oleosins of Brassica napus L. (oilseed rape) synthesized by in-vitro translation were found to be very efficiently targeted to microsomal membranes but only poorly translocated to oil bodies or emulsified oil. The use of other bilayer membranes as controls showed that this interaction was specific. The rate of oleosin synthesis in the presence of microsomes was enhanced about threefold, indicative of the involvement of the signal-recognition particle in the targeting process. There is no evidence for the cleavage of the protein during targeting and the protein sequence reveals no consensus cleavage site for the signal peptide. Protection experiments using Proteinase K revealed that about 6 kDa of the protein is exposed on the cytoplasmic side of the ER but the remainder is protected. Carbonate (pH 11) washing of microsomal membranes after in-vitro translation confirmed that oleosins have a domain which remains inserted in the ER rather than the protein being transported completely into the lumen of the ER. These results indicate that oleosins are transported via the ER prior to their accumulation on oil bodies.     

Starch metabolism in developing embryos of oilseed rape

The aim of this work was to characterise the metabolism of starch in developing embryos of oilseed rape (Brassica napus L. cv. Topaz). The accumulation of starch in embryos in siliques which were darkened or had been exposed to the light was similar, suggesting that the starch is synthesised from imported sucrose rather than via photosynthesis in the embryo. Starch content and the activities of plastidial enzymes required for synthesis of starch from glucose 6-phosphate (Glc6P) both peaked during the early-mid stage of cotyledon development (i.e. during the early part of oil accumulation) and then declined. The mature embryo contained almost no starch. The starch-degrading enzymes α-(EC 3.2.1.1) and β-amylase (EC 3.2.1.2) and phosphorylase (EC 2.4.1.1) were present throughout development. Most of the activity of these three enzymes was extraplastidial and therefore unlikely to be involved in starch degradation, but there were distinct plastidial and extraplastidial isoforms of all three enzymes. Activity gels indicated that distinct plastidial isoforms increase during the change from net synthesis to net degradation of starch. Plastids isolated from embryos at stages both before and after the maximum starch content could convert Glc6P to starch although the rate was lower at the later stage. The results are consistent with the idea that starch synthesis and degradation occur simultaneously during embryo development. The possible roles of transient starch accumulation during embryo development are discussed. Received: 15 May 1997 / Accepted: 30 May 1997     

The rough endoplasmic reticulum is the site of reserve-protein synthesis in developing Phaseolus vulgaris cotyledons

Cytyledons of the common bean, Phaseolus vulgaris L., were incubated with radioactive amino acids at different stages of seed development. The proteins were fractionated by ion-exchange chromatography, sucrose gradients, and sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis. From 16 to 28 d after flowering about 40% of the incorporated radioactivity was associated with the polypeptides of vicilin and 10% with those of phytohemagglutinin.Polysomes were isolated from developing cotyledons 20–25 d after flowering and free polysomes were separated from membrane-bound polysomes. Aurintricarboxylic acid, an inhibitor of initiation in cell-free translation systems, did not inhibit the incorporation of amino acids into in-vitro synthesized proteins, indicating that synthesis was limited to the completion of already initiated polypeptides. Autofluorography of SDS-polyacrylamide gels showed that the two classes of polysomes made two different sets of polypeptides and that there was little overlap between these two sets.Four polypeptides similar in size to the 4 polypeptides of vicilin were made by membrane-bound polysomes and not by free polysomes. Antibodies specific for vicilin bound to those 4 polypeptides. Free polysomes made only polypeptides which did not bind to antibodies specific for vicilin. Antibodies against phytohemagglutinin did not bind to any of the invitro synthesized polypeptides.The membranes to which the polysomes were bound were characterized on sucrose gradients and by electron microscopy. Polysomes recovered from membranes which banded on top of 35 and 50% sucrose synthesized the vicilin polypeptides most rapidly. These membrane fractions were rich in vesicles of rough endoplasmic reticulum (ER). The ER marker-enzyme NADH-cytochrome-c reductase banded with an average density of 1.18 g/cm³ (40% w/w sucrose) on continuous gradients. These experiments demonstrate that the ER is the site of vicilin synthesis in developing bean cotyledons. Quantitative determinations of several ER parameters (RNA and lipid-phosphate content, NADH-cytochrome-c-reductase activity) show that expansion of the cotyledons is accompanied by a 4-6-fold increase in ER.     

The Endoplasmic Reticulum of Mung Bean Cotyledons: ROLE IN THE ACCUMULATION OF HYDROLASES IN PROTEIN BODIES DURING SEEDLING GROWTH

The subcellular localization of two hydrolases (ribonuclease and vicilin peptidohydrolase) which are synthesized de novo in the cotyledons of mung bean seedlings was studied. Earlier experiments had shown that both enzymes accumulate in the protein bodies in the course of seedling growth. Two methods to fractionate subcellular organelles were used to demonstrate that a significant proportion of the enzymes is organelle-associated. This proportion is highest (up to 50% for vicilin peptidohydrolase and 15% for ribonuclease) when synthesis of the enzymes has just started. Evidence obtained with isopycnic sucrose gradients indicates that both hydrolases are associated with membranes rich in NADH-cytochrome c reductase, a marker enzyme for the endoplasmic reticulum (ER). The hydrolases band with the NADH-cytochrome c reductase under conditions where the ribosomes remain attached or are detached from the ER-derived vesicles. Treatment of the ER-derived vesicles with Triton X-100 shows that vicilin peptidohydrolase and vesicle membranes can be physically separated without dissolving the membranes, indicating that the proteinase is soluble within the vesicles. These data support the conclusion that the ER is involved in the transport of ribonuclease and proteinase to the protein bodies.     

The accumulation of triacylglycerols within the endoplasmic reticulum of developing seeds of Helianthus annuus

Microsomes isolated from developing seeds of Helianthus annuus were prepared in a medium which ensured that endoplasmic reticulum (ER)-bound polysomes remained attached to the ER during homogenization. The microsomes were then incubated with the substrates necessary to sustain the synthesis of triacylglycerols (TAGs). Microsomes that contained high activities of the enzymes involved in the synthesis of TAGs (the enzymes of the Kennedy pathway) accumulated TAGs synthesized in vitro , resulting in a decrease in their buoyant density. These light membrane fractions could therefore be separated on discontinuous sucrose density gradients from microsomes containing low activities of the enzymes of the Kennedy pathway. Analysis of the microsome fractions by ¹H-NMR spectroscopy showed that the TAGs synthesized in the microsomes in vitro were tumbling isotropically in an environment similar to that of the TAGs in oil bodies. Western blot analysis revealed that microsomes which synthesized large amounts of TAGs in vitro were also substantially enriched in oleosins. In addition, labelling studies indicated that the oleosins newly synthesized in vitro by ‘run-on' translation of ER-bound polysomes also localized to light membrane fractions. This indicates that oleosins are specifically enriched in regions of the ER involved in the biogenesis of the oil body.     

Subcellular fractionation of epiphyseal cartillage isolation of matrix vesicles and profiles of enzymes,phospholipids, calcium and phosphate

Epiphyseal cartilage was fractionated into subcellular components by non-enzymatic methods, and analyzed for activity of marker enzymes, for phospholipids, and for calcium and inorganic phosphate. Alkaline phosphatase, a marker enzyme for matrix vesicles and plasma membranes, was concentrated in the 100 000 × g (microsomal) pellet and, upon subsequent frationalism, in the low-density fractions from the sucrose gradient. Mitochondrial and endoplasmic reticular enzymes were localized primarily in the 20 000 × g pellet, lysosomal enzymes predominantly in the supernate from the microsomal pellet. Two phospholipids characteristic of matrix vesicles, sphingomyelin and phosphatidylserine, were enriched in the low-density sucrose fractions; however, unlike matrix vesicles, there was no depletion in phosphatidylcholine or increase in lysophospholipids. Ca and inorganic P were concentrated in the higher-density fractions, the amounts in the lower-density fractions being some- what lower than those seen in matrix vesicles. The alkaline phosphatase-rich, low-density fractions were thus not identical to matrix vesicles isolated by collagenase digestion, but rather appear to be composed primarily of plasma membranes. Enzyme profiles indicate they were relatively free of mitochondrial, endoplasmic reticular and lysosomal contaminants. The data further indicate that significant modification of the phospholipid, electrolyte, and possibly enzyme content of chondrocyte plasma membranes, must occur during blebbing and matrix vesicle formation.     

Fatty acid distribution and lipid metabolism in developing seeds of laurate-producing rape (Brassica napus L.)

The fatty acid composition and content of membrane and storage lipids of two transgenic laurate-producing rape (Brassica napus L.) lines were monitored during seed development. The two lines, the medium-laurate (ML) line and the high-laurate (HL) line, accumulated 34 mol% and 55 mol% of laurate in their seed triacylglycerols, respectively. The diacylglycerols contained about 17 and 33 mol% of laurate in the ML- and HL-lines, respectively, from the mid-stage of seed development up to seed maturity. The ML-line showed a maximal relative laurate content in phosphatidylcholine (17 mol%) at the mid-stage of seed development whereafter the content decreased to 2.7 mol% with seed maturity. The laurate content in phosphatidylcholine was observed to remain high (26 mol%) in the HL-line from the mid-stage to the end of triacylglycerol deposition. Thereafter, the relative content decreased and reached 6.6 mol% in the mature seeds. There was an enhanced activity of lauroyl-phosphatidylcholine- metabolizing enzymes in the seed membranes from laurate-producing lines compared with control lines, which might explain the decrease seen in laurate content in phosphatidylcholine during seed maturation. A comparison of the laurate distribution in the lipids from developing laurate-producing rape seeds and developing seeds from three species naturally accumulating laurate at similar levels revealed differences in laurate metabolism compared with these species. The results suggest that phospholipids and triacylglycerols are synthesized from the same diacylglycerol pool in rape seeds and that rape lysophosphatidic acid acyltransferase and diacylglycerol acyltransferase do not have the same preference for laurate substrates as the corresponding enzymes in seed tissues naturally accumulating this acyl group. In addition, the mechanisms that specifically remove or exclude laurate from membrane lipids appear less effective in rape seed than in tissues naturally evolved to synthesize laurate-rich oils. Received: 23 December 1996 / Accepted: 16 April 1997     

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.     

Osmotic potential and abscisic acid regulate triacylglycerol synthesis in developing wheat embryos

This work was carried out to determine what factors in the developing wheat (Triticum aestivum L.) grain are involved in regulating the metabolism of the triacylglycerol (TAG) storage reserves. When embryos are isolated from the grain and incubated in media for 4 d the TAG content is affected in three ways. In the basal medium (dilute buffer) the content falls; in 30 mM sucrose the content remains unchanged; in sucrose supplemented with an osmoticum (400 mM mannitol) or abscisic acid (1 M ABA) the TAG content increases. Effective osmotic potentials and ABA concentrations fit well with their respective values in planta. The fatty-acid composition of TAG accumulated in vitro is close to that in planta but in the absence of ABA or osmoticum there is a fall in the C₁₈C₁₆ ratio. Experiments with [¹⁴C] acetate show that the in-planta rate of incorporation into TAG can only occur in isolated embryos treated osmotically or with ABA, while there seems to be no effect of these two factors on TAG breakdown. An osmotic shock (dilute buffer) for only 2 h causes a rapid fall in TAG synthesis which continues for ca. 24 h after which it recovers. Abscisic acid protects against osmotic shock. It is concluded that TAG synthesis in developing wheat embryos is regulated by the osmotic potential and-or ABA, and that the embryos are very sensitive to short-term perturbations of these two factors.Abbreviations ABA abscisic acid - dpa days post anthesis - TAG triacylglycerol We are grateful to the European Economic Community for a Fellowship to R.R.S. which provided financial support for this work.     

PREPARATION OF PLASMA MEMBRANE FROM ISOLATED NEURONS

A bulk fraction enriched with respect to neuronal cell bodies was used as starting material for the isolation of neuronal plasma membrane The cells were gently homogenized in isotonic sucrose and a crude membrane containing fraction sedimented at 3000 g. Subsequently, the membrane fraction was purified on a discontinuous sucrose density gradient between 35% and 25 5% sucrose (w/w). Enzymatic analyses showed a 4–5-fold enrichment in plasma membrane markers, and a 10–15% contamination of mitochondrial and microsomal material. Electron micrographs of the membrane fraction confirmed the enzymatic data Fragmented membranes were found, mainly in vesicular form No ribosomes, but a few mitochondria and some multilamellar membranes were seen     

Modulation of membrane composition of swine vascular smooth muscle cells by homologous lipoproteins in culture

Swine vascular smooth muscle cells were exposed to homologous low-density or high-density lipoprotein fractions for 24 h. Total cell membranes were isolated from the post-nuclear supernatant of the cell homogenates, fractionated by sucrose density gradient centrifugation and characterized by enzyme assays. The membrane fraction with the lowest density was enriched in plasma membrane marker enzymes. Cholesterol analysis showed that cells exposed to low-density lipoprotein had higher cholesterol-to-protein ratios in total cells, total cell membranes and individual membrane fractions than had the cells exposed to high-density lipoproteins. Cholesterol-to-phospholipid ratios of the plasma membrane-enriched fraction from cells exposed to low-density lipoprotein were higher than the same membrane fraction of cells exposed to high-density lipoprotein. Studies with iodinated lipoproteins showed that these compositional changes could not be due to lipoprotein contamination. Membrane microviscosity was determined by fluorescence depolarization with diphenylhexatriene and the microviscosity of the plasma membrane-enriched fraction was different in the cells exposed to the two different lipoprotein fractions. This difference in membrane microviscosity was significant only when the medium cholesterol content was 40 μg per ml or greater; cells exposed to low-density lipoprotein gave membranes with higher microviscosity.These results demonstrate that the properties of vascular smooth muscle cell membranes are influenced by exposure of the cells to homologous lipoprotein fractions.     

Subcellular localization of glycosidases and glycosyltransferases involved in the processing of N-linked oligosaccharides

Using isopycnic sucrose gradients, we have ascertained the subcellular location of several enzymes involved in the processing of the N-linked oligosaccharides of glycoproteins in developing cotyledons of the common bean, Phaseolus vulgaris. All are localized in the endoplasmic reticulum (ER) or Golgi complex as determined by co-sedimentation with the ER marker, NADH-cytochrome c reductase, or the Golgi marker, glucan synthase I. Glucosidase activity, which removes glucose residues from Glc₃Man₉(GlcNAc)₂, was found exclusively in the ER. All other processing enzymes, which act subsequent to the glucose trimming steps, are associated with the Golgi. These include mannosidase I (removes 1-2 mannose residues from Man_6-9[GlcNAc]₂), mannosidase II (removes mannose residues from GlcNAcMan₅[GlcNAc]₂), and fucosyltransferase (transfers a fucose residue to the Asn-linked GlcNAc of appropriate glycans). We have previously reported the localization of two other glycan modifying enzymes (GlcNAc-transferase and xylosyltransferase activities) in the Golgi complex. Attempts at subfractionation of the Golgi fraction on shallow sucrose gradients yielded similar patterns of distribution for all the Golgi processing enzymes. Subfractionation on Percoll gradients resulted in two peaks of the Golgi marker enzyme inosine diphosphatase, whereas the glycan processing enzymes were all enriched in the peak of lower density. These results do not lend support to the hypothesis that N-linked oligosaccharide processing enzymes are associated with Golgi cisternae of different densities.     

Two novel diacylglycerol acyltransferase genes from Xanthoceras sorbifolia are responsible for its seed oil content

Xanthoceras sorbifolia is an excellent model system for studying triacylglycerol (TAG) biosynthesis in woody oilseed plants due to the high amount of seed oil, which is important for food and industrial uses. TAG is the major form of stored lipids in seeds and diacylglycerol acyltransferase (DGAT; EC 2. 3. 1. 20) catalyzes the final and critical step of TAG synthesis. Here, two novel DGAT genes, designated XsDGAT1 and XsDGAT2, were cloned from developing X. sorbifolia embryos. Sequence analysis showed that XsDGAT1 had little sequence homology to XsDGAT2. Heterologous expression of XsDGAT1 and XsDGAT2 in TAG-deficient yeast mutants restored TAG synthesis, confirming their biological activity. Expression of the two genes in wild-type Arabidopsis led to TAG synthesis and an increase in total seed oil in transgenic plants, with XsDGAT1 appearing to contribute to TAG synthesis at a greater level. Comparison of the expression patterns revealed that both XsDGAT1 and XsDGAT2 were expressed in the examined tissues and had similar spatiotemporal expression patterns with higher expression in embryos than in leaves and petals. The expression patterns of both XsDGAT1 and XsDGAT2 correlated with oil accumulation in developing X. sorbifolia embryos. These data suggest that XsDGAT1 and XsDGAT2 are both responsible for TAG synthesis in X. sorbifolia seeds.     

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.     

Convergence of the endocytic and lysosomal pathways in soybean protoplasts

Ultrastructural analysis of endocytosis of cationized ferritin (CF) has been combined with ultrastructural localization of acid phosphatases (AcPase) in soybean (Glycine max (L.) Merr.) protoplasts. While CF is an electron-dense marker of organelles of the endocytic pathway, ultrastructural histochemistry of AcPase identifies the organelles involved in the synthesis, transport, and storage of lytic-compartment enzymes, i.e. the lysosomal pathway. Acid phosphatases have been localized using both lead- and cerium-precipitation techniques. Protoplasts have been exposed to CF for 5 min, 30 min, or 3 h and processed for AcPase localization. At 5 min, smooth vesicles contain both CF and AcPase. By 30 min, Golgi cisternae and multivesicular bodies contain both labels. By 3 h, vacuoles become labelled with both CF and AcPase. The large central vacuoles contain intraluminal membranes which are associated with both AcPase and CF. These observations extend the analogy between plant vacuoles and animal lysosomes and demonstrate the points at which the endocytic pathway of plants converges with the lysosomal pathway.Abbreviations AcPase acid phosphatase - CF cationized ferritin - ER endoplasmic reticulum - MVB multivesicular body - PCR partially coated reticulum - PM plasma membrane     

Heat Shock Inhibits alpha-Amylase Synthesis in Barley Aleurone without Inhibiting the Activity of Endoplasmic Reticulum Marker Enzymes

The effects of heat shock on the synthesis of α-amylase and on the membranes of the endoplasmic reticulum (ER) of barley (Hordeum vulgare) aleurone were studied. Heat shock, imposed by raising the temperature of incubation from 25°C to 40°C for 3 hours, inhibits the accumulation of α-amylase and other proteins in the incubation medium of barley aleurone layers treated with gibberellic acid and Ca²⁺. When ER is isolated from heat-shocked aleurone layers, less newly synthesized α-amylase is found associated with this membrane system. ER membranes, as indicated by the activities of NADH cytochrome c reductase and ATP-dependent Ca²⁺ transport, are not destroyed by heat stress, however. Although heat shock did not reduce the activity of ER membrane marker enzymes, it altered the buoyant density of these membranes. Whereas ER from control tissue showed a peak of marker enzyme activity at 27% to 28% sucrose (1.113-1.120 grams per cubic centimeter), ER from heat-shocked tissue peaked at 30% to 32% sucrose (1.127-1.137 grams per cubic centimeter). The synthesis of a group of proteins designated as heat-shock proteins (HSPs) was stimulated by heat shock. These HSPs were localized to different compartments of the aleurone cell. Several proteins ranging from 15 to 30 kilodaltons were found in the ER and the mitochondrial/plasma membrane fractions of heat-shocked cells, but none of the HSPs accumulated in the incubation medium of heat-shocked aleurone layers.     

Synthesis of an integral protein of the protein-body membrane in Phaseolus vulgaris cotyledons

Protein-body membranes (PBMs) were isolated from cotyledons of Phaseolus vulgaris L. by a procedure involving osmotic shock of purified protein bodies. The purified PBMs have a characteristic density of 1.16 g cm^-3. Treatment of the membranes with increasing concentrations of detergent (Triton X-100) or with a solution at pH 12.0 showed that the membranes contained a characteristic integral protein (IMP) with a relative molecular mass of 25,000. This IMP is not a glycoprotein. When developing cotyledons were labeled with ³H-amino acids for 2–3 h, a radioactive polypeptide with the same mobility on denaturing polyacrylamide gels as IMP was found to be associated with the rough endoplasmic reticulum (ER). During a 24-h chase, a considerable portion of the radioactivity slowly transferred into the IMP associated with more rapidly sedimenting organelles, which sedimented in the same region of the sucrose gradients as the PBMs. Antibodies prepared against purified IMP crossreacted with an ER-associated protein which had the same mobility on denaturing acrylamide gels as authentic IMP. Synthesis of IMP occurred at all stages of cotyledon development examined, but not during seed germination. The results show that a newly synthesized protein of the PBM is associated with the rough ER, just like the soluble matrix proteins, phaseolin (R. Bollini, W. Van der Wilden and M.J. Chrispeels, 1983, J. Cell Biol. 96,999–1007) and phytohemagglutinin (M.J. Chrispeels and R. Bollini, 1982, Plant Physiol. 70, 1425–1428), but that the chase-out from the ER is much slower for IMP than for the matrix proteins.Abbreviations EDTA ethylenediamino-tetraacetic acid - ER endoplasmic reticulum - IMP integral membrane protein - PB protein body - PBM protein-body membrane - PHA phytohemagglutinin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Increased levels of glycerol-3-phosphate lead to a stimulation of flux into triacylglycerol synthesis after supplying glycerol to developing seeds of<Emphasis Type="Italic"> Brassica napus</Emphasis> L.<Emphasis Type="Italic"> in planta</Emphasis>

Glycerol-3-phosphate (glycerol-3P) is a primary substrate for triacylglycerol synthesis. In the present study, changes in the levels of glycerol-3P during rape (Brassica napus L.) seed development and the influence of manipulating glycerol-3P levels on triacylglycerol synthesis were investigated. (i) Glycerol-3P levels were high in young seeds and decreased during seed development at 30 and 40 days after flowering (DAF), when lipid accumulation was maximal. (ii) To manipulate glycerol-3P levels in planta, various concentrations of glycerol were injected directly into 30-DAF seeds, which remained otherwise intact within their siliques and attached to the plant. Injection of 0–10 nmol glycerol led to a progressive increase in seed glycerol-3P levels within 28 h. (iii). Increased levels of glycerol-3P were accompanied by an increase in the flux of injected [¹⁴C]sucrose into total lipids and triacylglycerol, whereas fluxes to organic acids, amino acids, starch, protein and cell walls were not affected. (iv) When [¹⁴C]acetate was injected into seeds, label incorporation into total lipids and triacylglycerol increased progressively with increasing glycerol-3P levels. (v) There was a strong correlation between the level of glycerol-3P and the incorporation of injected [¹⁴C]acetate and [¹⁴C]sucrose into triacylglycerol. (v) The results provide evidence that the prevailing levels of glycerol-3P co-limit triacylglycerol synthesis in developing rape seeds.Abbreviations DAF Days after flowering - DAG Diacylglycerol - G3PAT Glycerol-3-phosphate acyltransferase - Glycerol-3P Glycerol-3-phosphate - PA Phosphatidic acid - PC Phosphatidylcholine - TAG Triacylglycerol,     

Ultrastructural localization of cytoplasmic phosphatases in preimplantation mouse embryos.

The appearance and localization of the cytoplasmic phosphatases [acid phosphatase (AcPase) as a marker of lysosomes, TPPase as a marker of the Golgi apparatus, and NDPase (IDPase) as enzymatic marker of the endoplasmic reticulum (ER)] were cytochemically studied on the ultrastructural level in secondary oocytes and in preimplantation mouse embryos. The detectable AcPase activity, located on the inner surface of the membrane delimiting some cytoplasmic vacuoles (lysosomes and autophagic vacuoles), appears at the eight-cell stage and grows pregressively stronger up to the blastocyst stage. Golgi-associated reaction for TPPase was detectable in oocytes, dropped in one-cell embryos and became negative in the two-cell embryos. The reaction for TPPase and IDPase was present in plasma membranes of oocytes and early embryos and appeared in the delimiting membrane of some cytoplasmic vesicles in eight-cell embryos. Some activity of IDPase was found in small segments of the ER at the morula and blastocyst stage. The observed results suggest that the lysosomes are the first organelles in early embryos showing activity of the marker enzymes of the phosphatase type, while the activity of other marker enzymes is mainly concentrated in the plasma membrane of blastomeres. It cannot be excluded, however, that positive reaction for TPPase and IDPase in the plasma membrane results from nonspecific action of other phosphatases.     

Intracellular localization of phosphatidylcholine and phosphatidylethanolamine synthesis in cotyledons of cotton seedlings

Subfractionation of clarified cotyledon homogenates of cotton (Gossypium hirsutum L.) seedlings on sucrose gradients revealed a single coincident peak of cholinephosphotransferase (EC 2.7.8.2) (CPT) and ethanolaminephosphotransferase (EC 2.7.8.1) (EPT) activities, which equilibrated with the main peak of Antimycin A-insensitive NADH:cytochrome c reductase (CCR) activity. The small percentage of CPT and EPT activities (less than 5% of the total) in glyoxysome-enriched pellets equilibrated with cytochrome c oxidase activity, not with catalase activity. Preincubation of microsomes (containing 83% of total CPT and EPT activities) in 0.2 millimolar MgCl₂ followed by subfractionation on sucrose gradients resulted in peak CPT and EPT activities equilibrating with peak CCR activity at 24% (w/w) sucrose. Preincubation of microsomes with ¹⁴C-CDPcholine (or ¹⁴C-CDPethanolamine) resulted in synthesis and incorporation of ¹⁴C-phosphatidylcholine (PC) (or ¹⁴C-phosphatidylethanolamine, PE) into membranes at the same density. Increasing the Mg²⁺ concentration to 2.0 millimolar facilitated binding of ribosomes and caused a concomitant shift in density (to 34% w/w sucrose) of peak CPT, EPT, and CCR activities. Under these conditions, newly synthesized and incorporated ¹⁴C-PC (or PE) was recovered in these membranes. Transmission electron microscopy of this fraction confirmed binding of ribosomes to membranes. Radiolabeling in vivo of cotyledons with [methyl-¹⁴C] choline chloride or [1,2 ethanolamine-¹⁴C] ethanolamine hydrochloride resulted in a linear incorporation of radiolabel into PC or PE in a time dependent manner. Subfractionation of homogenates of radiolabeled cotyledons on sucrose gradients showed that membranes sedimenting at 24% (w/w) sucrose (ER) contained the majority of radiolabeled PC and PE with a minor peak at 40% (w/w) sucrose (mitochondria), but no radioactive PC or PE was recovered in glyoxysomes. These results indicate that ER in cotyledons of germinated cotton seedlings is the primary subcellular site of PC and PE synthesis. This is similar to the situation in endosperm tissue but distinctly different from root and hypocotyl tissue where Golgi are a major subcellular site of PC and PE synthesis.