Development of woronin bodies from microbodies inFusarium oxysporum f. sp.lycopersici

Summary Woronin bodies are cytoplasmic organelles which commonly lie near the septa in ascomycetous fungi. Although these organelles were observed nearly 100 years ago, little is known about their origin and development. The present ultrastructural investigation describes the ontogeny of Woronin bodies inFusarium oxysporum f. sp.lycopersici [Sacc.] Snyd. and Hans. In this fungus, Woronin bodies are produced by microbodies. Development of the Woronin body begins with the appearance of electron dense material within the microbody. This material aggregates adjacent to the membrane of the microbody and condenses into a single paracrystalline inclusion. Following its formation, the inclusion is gradually extruded and is eventually separated from the parent organelle by an exocytotic mechanism. After the separation, the paracrystalline inclusion is found at the septal pore. Although many recent electron microscopic studies have used various terms to designate these membrane bound organelles, inFusarium these inclusions are believed to correspond to the Woronin bodies initially described by light microscopists.     

Ultracytochemical localization of the vacuolar marker enzymes alkaline phosphatase,adenosine triphosphatase,carboxypeptidase Y and aminopeptidase reveal new concept of vacuole biogenesis in Saccharomyces cerevisiae

Summary Logarithmic cultures of Saccharomyces cerevisiae strains LBG H 1022, FL-100, X 2180 1A and 1B were studied together with the mutants pep4-3, sec18-1 and sec7-1. The necessary ultrastructural observations showed that, as a rule, juvenile vacuoles were formed de novo from perinuclear endoplasmic reticulum cisternae (ER) packed and inflated with electron-dense (polyanionic) matrix material. This process was disturbed solely in the sec18-1 mutant ander non-permissive conditions. The vacuolar marker enzymes adenosine triphosphatase (ATPase) and alkaline phosphohydrolase (ALPase) were assayed by the ultracytochemical cerium precipitation technique. The neutral ATPase was active in vacuolar membranes and in the previously shown (coated) microglobules nearby. ALPase activity was detected in microglobules inside juvenile vacuoles, inside nucleus and in the cytoplasm as well as in the membrane vesicles and in the periplasm. The sites of vacuolar protease carboxypeptidase Y (CPY) activity were assayed using N-CBZ-l-tyrosine-4-methoxy-2-naphthylamide (CBZ-Tyr-MNA) as substrate and sites of the aminopeptidase M activity using Leu-MNA as substrate. Hexazotized p-rosaniline served as a coupler for the primary reaction product of both the above proteases (MNA) and the resulting azo-dye was osmicated during postfixation. The CPY reaction product was found in both polar layers of vacuolar membranes (homologous to ER) and in ER membranes enclosing condensed lipoprotein bodies which were taken up by the vacuoles of late logarithmic yeast. Both before and after the uptake into the vacuoles the bodies contained the CPY reaction product in concentric layers or in cavities. Microglobules with CPY activity were also observed. Aminopeptidase was localized in microglobules inside the juvenile vacuoles. These findings combined with the previous cytochemical localizations of polyphosphates and X-prolyl-dipeptidyl (amino)peptidase in S. cerevisiae suggest the following cytologic mechanism for the biosynthetic protein transport: coated microglobules convey metabolites and enzymes either to the cell surface for secretion or enter the vacuoles in all phases of the cell cycle. The membrane vesicles represent an alternative secretory mechanism present in yeast cells only during budding. The homology of the ER with the vacuolar membranes and with the surface membranes of the lipoprotein condensates (bodies) indicates a cotranslational entry of the CPY into these membranes: The secondary transfer of a portion of CPY into vacuoles is probably mediated by the lipoprotein uptake process.     

ULTRASTRUCTURE OF THE SHOOT APEX OF TOMATO (LYCOPERSICON ESCULENTUM)

The vegetative shoot apical meristem of tomato (Lycopersicon esculentum Mill.) was examined at the ultrastructural level. The meristem consisted of a surface layer that was different from the rest of the meristem and was unique among the dicotyledonous species. The cells of the surface layer contained large distal vacuoles with relatively large electron-dense inclusions, proplastids with membrane-bound inclusions (MB), and differentiating chloroplasts. In addition, periclinal and oblique divisions were observed in the surface layer cells along with anticlinal divisions. The cells of the subsurface layers contained small vacuoles with fewer inclusions as well as proplastids of various shapes but without MB. Differentiating chloroplasts were not observed in these cells, but autophagic vacuoles at various stages of development were present. The normal complement of cell inclusions, e.g., the mitochondria, golgi bodies, endoplasmic reticulum (ER), ribosomes, and microtubules were observed in subsurface layers, and in many cells the ER was observed to be continuous with the outer membrane of the nuclear envelope and with the plasmalemma. Further below in the meristem, cells contained both the proplastids and differentiating chloroplasts with MB. In the latter, the outer membrane of the MB was found to be continuous with the developing lamellae, suggesting that MB probably serve as the storage centers for lamellae membranes. Near the base of the meristem, in the pith-rib meristem, enlarged cells containing large vacuoles and differentiated chloroplasts were present.     

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     

ELECTRON MICROSCOPY OF PHIALOCONIDIOGENESIS IN METARRHIZIUM ANISOPLIAE

Fine-structure observations with two different fixation procedures showed that phialide necks possessed a thickened electron-transparent wall layer. Phialoconidia developed from a wall layer which originated 1–1.5 μm within phialide necks. After conidium initials blew out of phialide tips and organelles entered, conidia were delimited by transverse septa which did not appear to be plugged by Woronin body-like plugs. Instead, septa appeared to become functionally complete by continued centripetal growth. Conidium-delimiting septa moved distally out of phialide necks as subsequent conidium initials formed. During this distal movement, septa increased in thickness and lamellae appeared on the conidium side; mature conidia had bipolarly lamellate cell walls. Conidial walls had a thin, ridged electron-dense outer wall layer and a thicker electron-transparent inner wall layer which increased in thickness centripetally after septum delimitation. Conidia were usually uninucleate and possessed conspicuous storage vacuoles with lipid and protein contents. Conidia also possessed numerous presumably lipid droplets. Multivesicular bodies were observed near conidium-delimiting septa and conidium walls which were increasing in thickness.     

Ultracytochemical localization of the vacuolar marker enzymes alkaline phosphatase, adenosine triphosphatase, carboxypeptidase Y and aminopeptidase reveal new concept of vacuole biogenesis in Saccharomyces cerevisiae

Logarithmic cultures of Saccharomyces cerevisiae strains LBG H 1022, FL-100, X 2180 1A and 1B were studied together with the mutants pep4-3, sec18-1 and sec7-1. The necessary ultrastructural observations showed that, as a rule, juvenile vacuoles were formed de novo from perinuclear endoplasmic reticulum cisternae (ER) packed and inflated with electron-dense (polyanionic) matrix material. This process was disturbed solely in the sec18-1 mutant under non-permissive conditions. The vacuolar marker enzymes adenosine triphosphatase (ATPase) and alkaline phosphohydrolase (ALPase) were assayed by the ultracytochemical cerium precipitation technique. The neutral ATPase was active in vacuolar membranes and in the previously shown (coated) microglobules nearby. ALPase activity was detected in microglobules inside juvenile vacuoles, inside nucleus and in the cytoplasm as well as in the membrane vesicles and in the periplasm. The sites of vacuolar protease carboxypeptidase Y (CPY) activity were assayed using N-CBZ-L-tyrosine-4-methoxy-2-naphthyl-amide (CBZ-Tyr-MNA) as substrate and sites of the amino-peptidase M activity using Leu-MNA as substrate. Hexazotized p-rosaniline served as a coupler for the primary reaction product of both the above proteases (MNA) and the resulting azo-dye was osmicated during postfixation. The CPY reaction product was found in both polar layers of vacuolar membranes (homologous to ER) and in ER membranes enclosing condensed lipoprotein bodies which were taken up by the vacuoles of late logarithmic yeast. Both before and after the uptake into the vacuoles the bodies contained the CPY reaction product in concentric layers or in cavities. Microglobules with CPY activity were also observed. Aminopeptidase was localized in microglobules inside the juvenile vacuoles. These findings combined with the previous cytochemical localizations of polyphosphates and X-prolyl-dipeptidyl (amino)peptidase in S. cerevisiae suggest the following cytologic mechanism for the biosynthetic protein transport: coated microglobules convey metabolites and enzymes either to the cell surface for secretion or enter the vacuoles in all phases of the cell cycle. The membrane vesicles represent an alternative secretory mechanism present in yeast cells only during budding. The homology of the ER with the vacuolar membranes and with the surface membranes of the lipoprotein condensates (bodies) indicates a cotranslational entry of the CPY into these membranes. The secondary transfer of a portion of CPY into vacuoles is probably mediated by the lipoprotein uptake process.     

Ultrastructure of the concentric membrane system in Arthrinium aureum

An ultrastructural study was performed on Arthrinium aureum. The fungi were treated with glutaraldehyde and osmium tetroxide fixation. The hypha and conidia has a concentric membrane system which consisted of multiple membranes of a myelinoid appearance, and continued to the conidia and hypha plasma membrane. The fungi were also treated with periodic acid-alkaline bismuth (PABi) staining after glutaraldehyde and osmium tetroxide fixation. PABi positive materials were found on the marginal glycogen granules, the concentric membrane system and the conidia plasma membrane.     

Observation of Woronin bodies in Arthrinium aureum by scanning electron microscopy

Woronin bodies are cytoplasmic organelles of filamentous fungi that can be observed on one, or both sides of each septum. The goal of this paper is to illustrate the presence of them in hyphae of Arthrinium aureum by means of scanning electron microscopy and to show that they act as a safety plug to close septa pores in hypha. Results show that Woronin bodies as an immediate response to prevent a cytoplasm loss. Results support hypothesis proposed previously in literature. This revised version was published online in June 2006 with corrections to the Cover Date.     

A proteomic approach towards the identification of the matrix protein content of the two types of microbodies in Neurospora crassa

Microbodies (peroxisomes) comprise a class of organelles with a similar biogenesis but remarkable biochemical heterogeneity. Here, we purified the two distinct microbody family members of filamentous fungi, glyoxysomes and Woronin bodies, from Neurospora crassa and analyzed their protein content by HPLC/ESI‐MS/MS. In the purified Woronin bodies, we unambiguously identified only hexagonal 1 (HEX1), suggesting that the matrix is probably exclusively filled with the HEX1 hexagonal crystal. The proteomic analysis of highly purified glyoxysomes allowed the identification of 191 proteins. Among them were 16 proteins with a peroxisomal targeting signal type 1 (PTS1) and three with a PTS2. The collection also contained the previously described N. crassa glyoxysomal matrix proteins FOX2 and ICL1 that lack a typical PTS. Three PTS1 proteins were identified that likely represent the long sought glyoxysomal acyl‐CoA dehydrogenases of filamentous fungi. Two of them were demonstrated by subcellular localization studies to be indeed glyoxysomal. Furthermore, two PTS proteins were identified that are suggested to be involved in the detoxification of nitroalkanes. Since the glyoxysomal localization was experimentally demonstrated for one of these enzymes, a new biochemical reaction is expected to be associated with microbody function.     

The effects of abscisic acid on the maturation ofBrassica napus somatic embryos

Summary A comparison of embryos, cultured for increasing periods of time with and without abscisic acid (ABA), was undertaken to investigate, at the ultrastructural level, the influence of this growth regulator on the maturation of rapeseed (Brassica napus) somatic embryos. In the absence of ABA, the embryos germinated precociously while lipid bodies (LB), which were not numerous, soon degraded, as revealed by a depletion process associated with the appearance of morphologically mature glyoxysomes and an increase in the number of mitochondria. Moreover, a lack of protein bodies indicated that storage protein accumulation was not initiated under these conditions. On the contrary, the addition of ABA (10 M) induced marked modification of embryo metabolism. Indeed, ABA completely prevented precocious embryo germination and inhibited lipid reserve catabolism. Moreover, the formation of small vacuoles and proliferation of rough endoplasmic reticulum in their vicinity suggested the onset of storage protein accumulation. After 15 days in the presence of ABA, the embryos contained abundant lipid and protein bodies. Nevertheless, these somatic embryos were not exactly the same as their mature zygotic counterparts since differences were found in chloroplasts, amyloplasts, and nuclear structures. These observations suggest that additional factors might be required to obtain fully mature somatic embryos.Abbreviations ABA abscisic acid - ABM ABA medium - BM basal medium - LB lipid bodies - MS Murashige and Skoog (1962) - PB protein bodies - RER rough endoplasmic reticulum     

ENDOSPERM STRUCTURE AND STORAGE RESERVE HISTOCHEMISTRY IN THE PALM,WASHINGTONIA FILIFERA

The endosperm of Washingtonia filifera consists of living cells with the same general cellular structure throughout the seed. The major storage reserves are carbohydrate, stored in the form of thickened walls; lipid, stored as numerous small lipid bodies which fill the cytoplasm; and protein, stored as large, but variably-sized, protein bodies. The protein bodies contain two types of inclusions: prismatically-shaped denser protein crystalloids and small crystalline deposits presumed to be phytic acid. The X-ray microanalysis shows these crystalline inclusions do contain P, Ca, Mg, and Fe. Protein bodies are positively stained with PAS. Nuclei are present in all cells, but stain very palely. Plastids and mitochondria are present, but infrequently seen. The plastids have few, poorly developed membranes. Endoplsasmic reticulum and dictyosomes are lacking. The cell wall is thick except in areas of pit fields and consists of three layers which differ in their staining with toluidine blue and in their ultrastructural characteristics: middle lamella, thickened outer wall, and thin inner wall. All wall layers are positively stained with PAS and calcofluor. Although general structural features of the endosperm in Washingtonia filifera are similar to those in date seeds, the composition of the wall polysaccharides and protein bodies appear to differ somewhat.     

Wheat (Triticum aestivum L.) [gamma]-Gliadin Accumulates in Dense Protein Bodies within the Endoplasmic Reticulum of Yeast

Following their sequestration into the endoplasmic reticulum (ER), wheat storage proteins may either be retained and packaged into protein bodies within this organelle or transported via the Golgi to vacuoles. We attempted to study the processes of transport and packaging of wheat storage proteins using the heterologous expression system of yeast. A wild-type wheat [gamma]-gliadin, expressed in the yeast cells, accumulated mostly within the ER and was deposited in protein bodies with similar density to natural protein bodies from wheat endosperm. This suggested that wheat storage proteins contain sufficient information to initiate the formation of protein bodies in the ER of a heterologous system. Only a small amount of the [gamma]-gliadin was transported to the yeast vacuoles. When a deletion mutant of the [gamma]-gliadin, lacking the entire N-terminal repetitive region, was expressed in the yeast cells, the mutant was unable to initiate the formation of protein bodies within the ER and was completely transported to the yeast vacuole. This strongly indicated that the information for packaging into dense protein bodies within the ER resides in the N-terminal repetitive region of the [gamma]-gliadin. The advantage of using yeast to identify the signals and mechanisms controlling the transport of wheat storage proteins and their deposition in protein bodies is discussed.     

Ultracytochemical localization of dihydroorotate dehydrogenase in mitochondria and vacuoles ofSaccharomyces cerevisiae

The coenzyme-independent dihydroorotate dehydrogenase (EC 1.3.3.1) linking the pyrimidine biosynthetic pathway to the respiratory chain, was ultracytochemically localized by the tetrazolium method in derepressed exponential-phase cultures ofSaccharomyces cerevisiae. Biochemical analysis showed a considerable variation of this enzyme activity in inverse proportion to the aeration of the yeast cultures. The assay also showed that after prefixation of yeast cells with 1% glutaraldehyde at 0°C for 20 min, approximately one-half of the enzyme activity was preserved. The cytochemical reaction mixture contained dihydroorotate (2 mmol/L), thiocarbamyl nitroblue tetrazolium (0.44 mmol/L), phenazine methosulfate (0.16 mmol/L) and KCN (1.7 mmol/L) in Tris-HCl buffer (100 mmol/L) of pH 8.0. The osmicated formazan deposits featured envelopes of mitochondria and of nuclei and were prominent in the mitochondrial inclusions and in the vacuolar membranes. The latter sites of dihydroorotate dehydrogenase activity represent biosynthetic activity in yeast vacuoles, still generally assumed to function as yeast lysosomes and storage organelles. In the light of the generally observed invasions of juvenile yeast vacuoles into mitochondria, the enzymic sites observed in mitochondrial inclusion were considered as evidence of the interactions of yeast vacuoles and mitochondria. Transfer of vacuolar membranes with dihydroorotate dehydrogenase activity into mitochondrial matrix is suggested.     

Lung alveolar cell cytosomes: A consideration of their significance

Summary An ultrastructural comparison of mammalian, reptilian, and amphibian lung alveolar cells, and avian lung atrial cells reveals that morphologically similar cytoplasmic bodies (cytosomes) occur in these cells. The cytosomes, which appear generally as osmiophilic, lamellae-containing, membrane-bound, round bodies 0.3 to 0.5 in diameter, are also similar to bodies occurring in epithelial cells of both physoclistous and physostomatous swimbladders of fishes. Because the function of both lung alveolar and swimbladder epithelial cells is gas-handling, the possibility is raised that the morphologically similar lamellae-containing bodies of these vertebrate cells are functionally identical. One function, suggested by other investigators, is that, in mammalian lungs, these bodies supply a surface-tension lowering material (surfactant). Because several assumptions concerning this proposed function remain unproved, an alternative proposal is speculatively explored. The suggestion is offered that cytosomes contain an antioxidant needed to protect alveolar and swimbladder cells against the toxic effects of the relatively high concentration of oxygen to which these cells are exposed.Supported by a research grant from the American Cancer Society, Oregon Division, Inc.     

The meiospore ofPhysoderma maydis. The causal agent ofPhysoderma disease of maize

Summary The meiospore ofPhysoderma maydis (Phycomycetes, Chytridiales, Physodermataceae) has a nuclear cap enclosing the cellular ribosomes within a double membrane, and double membranes traversing the nuclear cap. Aggregates of ribosomes not incorporated into the nuclear cap are also enclosed by double membranes. A vesicular network is observed in the anterior portion of the spore in direct connection with the nuclear cap membrane and with a stacked parallel array of membranes, which itself is connected with the nuclear cap membrane.The meiospore ofP. maydis contains a side body complex of the type observed in spores of theBlastocladiales. Vesicles enclose the side body complex and these vesicles are connected to the nuclear cap membrane and the nuclear envelope, and form a network which partially encloses the kinetosomal apparatus.The nuclear cap membrane, stacked array of membranes, and the vesicles which surround the side body complex and the kinetosomal apparatus contain an electron-dense amorphous material. On the basis of their ultrastructural appearance, these membranes are interpreted as part of a highly divided microbody.The ultrastructural organization of the meiospore ofP. maydis is compared to the structural organization observed in spores of theChytridiales, Blastocladiales, Monoblepharidales, andHarpochytriales. It is concluded that the structural organization of the meiospores ofP. maydis is the same as observed for members of theBlastocladiales, and it is suggested that thePhysodermataceae should be transferred from theChytridiales to theBlastocladiales.     

Woronin bodies,their impact on stress resistance and virulence of the pathogenic mould Aspergillus fumigatus and their anchoring at the septal pore of filamentous Ascomycota

Hyphae of filamentous Ascomycota consist of compartments that are connected via septal pores. To avoid a dramatic loss of cellular content after wounding, fungi developed mechanisms to occlude their septal pores. In most Pezizomycotina, so‐called Woronin bodies are anchored in proximity to the pore. This is a prominent example for precise spatial positioning of organelles, but so far the underlying molecular organization has remained largely unknown. Using the pathogenic mould Aspergillus fumigatus, we provide evidence that Woronin bodies are important for stress resistance and virulence. Furthermore the molecular machinery anchoring them at the septum is described. Namely, we have identified Lah as the tethering protein and provide evidence that the Woronin body protein HexA binds to the septal pore in a Lah‐dependent manner. Moreover, we demonstrate that a striking poly‐histidine motif targets HexA to the septal cell wall. Thus, the axis HexA‐Lah is an excellent candidate for the tether linking Woronin bodies to the septum. This model applies to A. fumigatus, but most likely also to the vast majority of the Pezizomycotina. Our findings shed light on the evolution of Woronin body anchoring and provide a basis for the development of novel strategies to combat fungal pathogens like A. fumigatus.     

Hyphal heterogeneity in Aspergillus oryzae is the result of dynamic closure of septa by Woronin bodies

Hyphae of higher fungi are compartmentalized by septa. These septa contain a central pore that allows for inter‐compartmental and inter‐hyphal cytoplasmic streaming. The cytoplasm within the mycelium is therefore considered to be a continuous system. In this study, however, we demonstrate by laser dissection that 40% of the apical septa of exploring hyphae of Aspergillus oryzae are closed. Closure of septa correlated with the presence of a peroxisome‐derived organelle, known as Woronin body, near the septal pore. The location of Woronin bodies in the hyphae was dynamic and, as a result, plugging of the septal pore was reversible. Septal plugging was abolished in a ΔAohex1 strain that cannot form Woronin bodies. Notably, hyphal heterogeneity was also affected in the ΔAohex1 strain. Wild‐type strains of A. oryzae showed heterogeneous distribution of GFP between neighbouring hyphae at the outer part of the colony when the reporter was expressed from the promoter of the glucoamylase gene glaA or the α‐glucuronidase gene aguA. In contrast, GFP fluorescence showed a normal distribution in the case of the ΔAohex1 strain. Taken together, it is concluded that Woronin bodies maintain hyphal heterogeneity in a fungal mycelium by impeding cytoplasmic continuity.     

Ultrastructural changes of renal tubules in the riboflavin deficient mouse

Ultrastructural changes of the tubular epithelium in the mouse kidney produced by dietary riboflavin deficiency were studied by electron microscopy and cytochemistry. In riboflavin deficient mouse kidney, the ultrastructural changes are localized to the pars recta of the proximal tubule. They comprise so called vacuolar degeneration on light microscopy, which consists of the formation of giant mitochondria and vacuoles. During the development of riboflavin deficiency, mitochondria decrease in number and enlarge in size through fusion. Sometimes they are larger than nuclei in size. The vacuoles observed in tubular epithelia are divided into two different groups according to their morphological characteristics and origins. One is derived from proliferated peroxisomes, and another from increased cytoplasmic bodies termed cytosomes and cytosegresomes. These increased vacuoles occupy almost all of cytoplasm. Cytochemical studies also reveal that these vacuoles are peroxisomes and lysosomes. These changes are reversible on supplementation with riboflavin.     

Heterogeneity of the endoplasmic reticulum with respect to lipid synthesis in developing seeds of Brassica napus L.

Studies of the sub-cellular location of storage triacylglycerol (TAG) synthesis in developing embryos of oilseed rape (Brassica napus L.) show that there is heterogeneity of the endoplasmic reticulum (ER) with respect to the enzymes of lipid synthesis. The enzymes of TAG synthesis were detected in two membrane fractions (equilibrium densities 1.05 and 1.10 g· ml^?1) isolated by sucrose-density-gradient centrifugation of homogenates from developing rape embryos. The synthesis of TAG by the lowdensity membranes has not been reported previously and was found in this study because the sucrose density gradients began at only 10% (w/w) sucrose. The pattern of activity of the enzymes involved in the synthesis of TAG in the higher-density fraction closely matched the marker enzymes for the ER; lyso-phosphatidylcholine acyltransferase and cytidine diphosphate-choline:diacylglycerol cholinephosphotransferase. The activity of the ER marker enzymes in the low-density membrane fraction, however, was very much lower when compared to those involved in the synthesis of TAG. Analysis of the lipids extracted from the low-density fraction revealed it contained about 50 mol% TAG compared with 15 mol% in the bulk ER, which may account for the low density of the membranes in this fraction. The possibility that the low-density membranes were the result of contamination of ER by oil bodies was ruled out by the use of oleosins as a marker for oil bodies. It is suggested that the low-density membranes are derived from a domain of the ER which is involved in the formation and secretion of TAG.     

Formation of protein storage bodies during embryogenesis in cotyledons of Sinapis alba L.

An electron microscopic investigation of fine structural changes in post-meristematic cotyledon mesophyll cells during the period of storage protein accumulation (16–32 d after pollination) showed that the rough ER, the Golgi apparatus and the developing vacuome are intimately involved in the formation of storage protein bodies (aleurone bodies). At the onset of storage protein accumulation (16–18 d after pollination) storage protein-like material appears within Golgi vesicles and preformed vacuoles. At a later stage (24 d after pollination) similar material can also be detected within vesicles formed directly by the rough endoplasmic reticulum (ER). It is concluded that there are two routes for storage protein transport from its site of synthesis at the ER to its site of accumulation in the vacuome. The first route involves the participation of dictyosomes while the second route bypasses the Golgi apparatus. It appears that the normal pathways of membrane flow in the development of central vacuoles in post-meristematic cells are used to deposit the storage protein within the protein bodies. Thus, the protein body can be regarded as a transient stage in the process of vacuome development of these storage cells.Abbreviation ER endoplasmic reticulum