Endoplasmic reticulum in developing seeds of Vicia faba

The changes in endoplasmic reticulum (ER) morphology during seed development have been followed using a thick section electron microscope technique. The tissues were stained with a zinc iodineosmium tetroxide complex which preferentially accumulated in the lumen between double membranes. Sections up to 2 m in thickness were examined in a high voltage electron microscope (HVEM) with tilt facility to produce stereo pairs. The micrographs from HVEM showed an increase in the extent of interconnecting tubular and cisternal ER during the protein deposition phase of seed maturation with subsequent degeneration of the cisternae to a reticular form during the final seed maturation phase. No evidence of cisternal ER vesicles was found, instead our work suggests that such structures are artefacts of thin sectioning with the so-called vesicles representing the interconnection of cisternal and tubular ER. The results are discussed with reference to the transport of storage protein from its site of synthesis, the rough cisternal ER, to that of accumulation, the vacuolar protein bodies.Abbreviations ER endoplasmic reticulum - HVEM high voltage electron microscopy     

Connections between dictyosomes,ER and GERL in cotyledons of mung bean (Vigna radiata L.)

Summary The connections and structural inter-relations of dictyosomes and endoplasmic reticulum (ER) in cotyledons of germinating mung beans were studied using thick (0.3 m) sections of aldehyde fixed, zinc iodide-osmium tetroxide (ZIO) impregnated tissue. The sections were examined by conventional (100 kV), rather than high voltage, transmission electron microscopy.Continuity of cisternal ER with tubular ER was confirmed and a direct connection of tubular ER totrans dictyosome cisternae was observed as were GERL networks associated withtrans dictyosome cisternae.Dictyosomes also gave rise to an extensive system of very fine tubules (10–20 nm diam) which have not been described previously in plant tissue. These tubules, which originated at thetrans dictyosome face, extended throughout the cytoplasm and were found connected to cisternal ER and tubular ER.The implications of these observations are discussed with regard to present ideas concerning endomembrane flow and protein sorting by the Golgi apparatus.     

The endoplasmic reticulum of mung-bean cotyledons: Quantitative morphology of cisternal and tubular ER during seedling growth

The ultrastructure of the endoplasmic reticulum (ER) in storage parenchyma cells in the cotyledons of mung beans (Vigna radiata L.) was examined during germination and seedling growth. Two different methods were used to visualize the ER: thin (0.08 m) sections of tissue fixed in formaldehyde and glutaraldehyde and post-fixed with osmium tetroxide, and thick (1 m) sections of tissue fixed in buffered aldehyde and post-fixed with zinc iodide-osmium tetroxide (ZIO). Changes in relative amounts of ER were quantified by morphometry (stereology).The ER occurs in two forms: a cisternal form with associated ribosomes which can be seen at all stages from imbibition to cotyledon senescence, and a tubular form which initially has associated ribosomes. Stereoscopic images of thick sections of cotyledons of 2-day-old seedlings show that the tubular ER consists of a three-dimensional array of interconnecting tubules which have numerous connections with the cisternal ER. The network of tubules and cisternae extends throughout the cytoplasm enveloping the protein bodies. Germination and seedling growth are accompanied by a reduction in the total volume occupied by the ER. This reduction is the result of a preferential loss of tubular ER and occurs largely before protein mobilization. Cisternal ER decreases during the first 48 h of imbibition and seedling growth, but storage cells subsequently show an increase in cisternal ER just prior to and during the period of protein mobilization. Cisternal ER remains conspicuous during the last phase of reserve mobilization when starch is broken down and the cells are starting autophagy.Abbreviations ER endoplasmic reticulum - ZIO zinc iodide-osmium tetroxide This is the second in a series of papers on the endoplasmic reticulum of mung bean cotyledons. The first paper is referenced herein as Gilkes and Chrispeels (1980)     

Protein accumulation in developing endosperm of a high-protein line of Triticum dicoccoides

Abstract. Endosperm tissue from developing grains of a line of wheat ( Triticum dicoccoides ) which accumulates up to 30% protein in the mature grain, was examined by electron microscopy to establish the ontogeny of the storage protein bodies. Ultrastructural evidence suggests that storage proteins of wheat may be transported from their site of synthesis on the rough endoplasmic reticulum (ER) to protein bodies by two different routes within the endomembrane system. The first route, which probably functions throughout protein deposition, involves the transport of protein from the cisternal rough ER to the protein vacuoles via the Golgi apparatus. The second route, observed 20 d after anthesis, appears to lead directly from dilated regions of the rough ER to protein vacuoles, bypassing the dictyosomes. Phytin inclusions are found in protein vacuoles of starchy endosperm cells adjacent to the aleurone layer of developing grain.     

Rice protein-body formation: all types are initiated by dilation of the endoplasmic reticulum

The ultrastructure of protein deposition in the starchy endosperm of developing rice (Oryza sativa L.) grains was examined in conventionally fixed (glutaraldehyde and osmium tetroxide) tissues and also in thick sections (0.3 m) of zinc iodide-osmium tetroxide post-fixed tissue. Three types of previously characterised protein body were observed and it was shown that each type was initiated by dilations of the endoplasmic reticulum. Crystalline type protein bodies were initiated by a ribosome-free dilation from rough cisternal endoplasmic reticulum and developed by inclusion of protein from dictyosome-derived vesicles. The large spherical and small spherical protein bodies developed within the cisternae of the rough endoplasmic reticulum.Abbreviations Cr crystalline protein body - DAF days after fertilization - ER endoplasmic reticulum - Ls large spherical protein body - Ss small spherical protein body - ZIO zinc iodide-osmium tetroxide     

Structure,differentiation, and multiplication of Golgi apparatus in fungal hyphae

Summary Golgi apparatus in subapical regions of hyphae consist of paranuclear dictyosomes with 4–5 cisternae each. Transverse and tangential sections provide ultrastructural evidence for a three-dimensional architectural model of the Golgi apparatus and a stepwise mechanism for dictyosome multiplication. The dictyosomes are polarized, with progressive morphological and developmental differentiation of cisternae from the cis to the trans pole. Small membrane blebs and transition vesicles provide developmental continuity between the nuclear envelope and the adjacent dictyosome cisterna at the cis face. Cisternae are formed as fenestrated plates with extended tubular peripheries. The morphology of each cisterna depends on its position in the stack, consistent with a developmental gradient of progressive maturation and turnover of cisternae. Mature cisternae at the trans face are dissociated to produce spheroid and tubular vesicles. Evidence in support of a schematic sequence for increasing the numbers of dictyosomes comes from images of distinctive and unusual forms of Golgi apparatus in hyphal regions where nuclei and dictyosomes multiply, as follows: (a) The area of the nuclear envelope exhibiting forming-face activity next to a dictyosome expands, which in turn increases the size of cisternae subsequently assembled at the cis face of the dictyosome. (b) As subsequent large cisternae are formed and mature as they pass through the dictyosome, an entire dictyosome about twice normal size is built up. The number of cisternae per stack remains the same because of continuing turnover and loss of cisternae at the trans face, (c) This enlarged dictyosome becomes separated into two by a small region of the nuclear envelope next to the cis face that acquires polyribosomes and no longer generates transition vesicles, (d) As a consequence, assembly of new dictyosomes is physically separated into two adjacent regions, (e) As.the enlarged cisternae are lost to vesiculation at the trans pole, they are replaced by two separate stacks of cisternae with typical normal diameters, (f) The net result is two adjacent dictyosomes where one existed previously. Dictyosome multiplication is thus accomplished as part of the normal developmental turnover of cisternae, without interrupting the functioning of the Golgi apparatus as it continues to produce new secretory vesicles from mature cisternae at the trans face. Coordination of Golgi apparatus multiplication with nuclear division ensures that each daughter nucleus receives a complement of paranuclear dictyosomes.     

Membranfluß und Nucleosiddiphosphatase-Reaktion in Wurzelhaaren von Lepidium sativum

Summary Root hairs of Lepidium sativum were incubated with a Wachstein-Meisel medium in experiments designed to localize the activity of nucleoside diphosphatase(s). Electron dense precipitates were found in the ER and in Golgi cisternae of the secretory face of the dictyosomes and their adjacent Golgi vesicles. Such precipitates were absent in the Golgi cisternae of the regeneration face of the dictyosomes and in the detached Golgi vesicles which extrude pectic cell wall substances. These results may be the consequence of the normal cycle of membrane compounds associated with the secretion in which the nucleoside diphosphatase(s) participate (by activation and inactivation) as one of the cycling components. Alternatively the nucleoside diphosphatase(s) may undergo a special cycle in which they are transferred from one cisterna or its vesicles to the next as part of the process of cisternal maturation.     

Pentachlorophenol affects mitochondria and induces formation of golgi apparatus-endoplasmic reticulum hybrids in tobacco pollen tubes

Summary The toxic effect of pentachlorophenol (PCP) on the growth and ultrastructure of tobacco pollen tubes was tested using a semivivo technique of tube culture. In this technique the pollen tubes were allowed to grow in the pistilin situ for 24 hr before they protruded from the cut end of the style and came into contact with the medium containing PCP. The inhibitory effect of different PCP concentrations was determined by measuring the length of tube bundles. The intracellular action of PCP was analysed by electron microscopy. This biocide caused four obvious alterations in the pollen tube ultrastructure: (1) swelling of the mitochondrial saccules; (2) enlargement of the dictyosomes by the increase of the cisternal diameter and the number of cisternae per stack; (3) formation of cup-shaped Golgi apparatus-endoplasmic reticulum hybrid structures (GER hybrids) showing continuities of ER and Golgi cisternae; (4) formation of stacked and/or concentric arrangements of rough ER cisternae. It is suggested that swelling of saccules was directly due to the uncoupling of oxidative phosphorylation whereas the changes of the endomembrane system were caused by energy depletion due to the inhibition of ATP synthesis. These changes are consistant with dynamic concepts of dictyosome and ER function when membrane formation exceeds membrane use in the production of secretory and transition vesicles. Thus, the enlargement of the dictyosomes and the formation of GER hybrids are thought to result from inhibition of budding of vesicles from the Golgi apparatus or from both the ER and the Golgi apparatus, respectively.     

Nuclear pore distribution and relation to adjacent cytoplasmic organelles in cotyledon cells of developing Vicia faba

Following a zinc iodine-osmium tetroxide fixation, nuclear pore distribution was studied in 0.3-m sections from cotyledons of developing Vicia faba L. Localised absence of nuclear pores was found to be associated with proximity of organelles to the nucleus. Golgi cisternae and mitochondria are associated with areas of pore absence while cisternal endoplasmic reticulum and tubular endoplasmic reticulum are linked with areas showing reduction in pore density. Pores were seen in the nuclear membrane adjacent to vacuoles. Pattern analysis of pore distribution indicated possible clustering within an overall regularity.Abbreviations ER endoplasmic reticulum - ZIO zinc iodine-osmium tetroxide     

Structure of amyloplasts and endoplasmic reticulum in the root caps of Lepidium sativum and Zea mays observed after selective membrane staining and by high-voltage electron microscopy

The structure of plastids in the root cap of cress and maize was studied by low- and high-voltage electron microscopy after staining their membranes with a mixture of zinc iodide and osmium tetroxide. In plastids of both species electron-opaque membranes were found in the plastid interior while membranes of lesser electron-opacity comprised the outer envelope and vesicles and cisternae underlying it. Electron-opaque tubules, often in groups attached to the inner membrane of the amyloplast envelope, were found in cress but not in maize. The internal, less-opaque membranes were often found associated with the starch grains. No specific association could be seen between amyloplasts and endoplasmic reticulum (ER); their surfaces showed no regular contact or connexion, though the amyloplasts clearly indented the underlying ER. The ER in statocytes was predominantly tubular in cress but predominantly cisternal in maize.Abbreviations ER endoplasmic reticulum - ZIO zinc iodideosmium tetroxide     

Immunocytochemical localization of reserve protein in the endoplasmic reticulum of developing bean (Phaseolus vulgaris) cotyledons

The ultrastructure of the storage parenchyma cells of the cotyledons of developing bean (Phaseolus vulgaris L.) seeds was examined in ultrathin frozen sections of specimens fixed in a mixture of glutaraldehyde, formaldehyde and acrolein, infused with 1 M sucrose, and sectioned at-80° C. Ultrastructural preservation was excellent and the various subcellular organelles could readily be identified in sections which had been stained with uranyl acetate and embedded in Carbowax and methylcellulose. The cells contained large protein bodies, numerous long endoplasmic reticulum cisternae, mitochondria, dictyosomes, and electron-dense vesicles ranging in size from 0.2 to 1.0 m. Indirect immunolabelling using rabbit immunoglobulin G against purified phaseolin (7S reserve protein), and ferritin-conjugated goat immunoglobulin G against rabbit immunoglobulin G was used to localize phaseolin. With a concentration of 0.1 mg/ml of anti-phaseolin immunoglobin G, heavy labeling with ferritin particles was observed ober the protein bodies, the cisternae of the endoplasmic reticulum, and the vesicles. The same structures were lightly labeled when the concentration of the primary antigen was 0.02 mg/ml. Ferritin particles were also found over the Golgi bodies. The absence of ferritin particles from other organelles such as mitochondria and from areas of cytoplasm devoid of organelles indicated the specificity of the staining, especially at the lower concentration of anti-phaseolin immunoglobulin G.Abbreviations ER endoplasmic reticulum - IgG immunoglobulin G     

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     

Acid phosphatase activity during spore differentiation of the red algaeGigartina teedii andChondria tenuissima

The acid phosphatase activity during carposporogenesis inGigartina and tetrasporogenesis inChondria was studied using the Gomori technique. During the first steps of gonimoblast maturation ofGigartina, portions of cytoplasm are ensheathed by ER cisternae with acid phosphatase activity, giving rise to autolysosomal concentric membrane bodies. In a similar way large mucilage sacs are severed. They extrude their contents in a kind of exocytosis. Multivesicular bodies, concentrically arranged cisternae and extracytoplasmic compartments, each with acid phosphatase activity, remain in young carpospores for some time, probably as remnants of the autophagocytotic and exocytotic events. The Golgi apparatus is poorly developed in gonimoblast cells and young carpospores. It becomes a prominent cell component in maturing carpospores and then participates in cell wall formation. Only some of the dictyosomal cisternae contain acid phosphatase; these are irregularly distributed in the dictyosome. — In pre- and postmeiotic tetraspore mother cells ofChondria massive lead deposits are found in the dictyosomes and in adjacent Golgi vesicles. Finer lead precipitates occur in ER cisternae, especially in those which are sequestering starch-grain-containing portions of the cytoplasm to give rise to autolysosomes. During cell cleavage, the dictyosomes aggregate. They become devoid of acid phosphatase activity with the exception of vesicles at the trans face. Later, Golgi stacks associate and have common, Gomori positively reacting, narrow cisternae at the cis face. The Golgi apparatus derived cored vesicles do not contain lead precipitates whereas the Golgi cisternae in the final stage of tetrasporogenesis show acid phosphatase activity. Variations in acid phosphatase distribution are explained in the light of current models of membrane flow.Dedicated to Univ.-Prof. DrO. Härtel on the occasion of his 80th birthday.     

A novel tubular array associated with protein bodies in the rough endoplasmic reticulum ofopaque-2 maize

Summary The seed storage proteins of maize (Zea mays L.) are synthesized during endosperm development on membrane-bound polyribosomes. Protein body formation in normal genotypes occurs via a sequential deposition of the various types of zeins, and leads to the formation of spherical structures with a diameter of about l m. In the endosperm mutantopaque-2 the level of one zein class is reduced; these kernels exhibit an opaque phenotype instead of the vitreous phenotype displayed in normal genotypes, presumably due to the decrease in total zein protein at the time of desiccation. Previous microscopic examination ofopaque-2 protein bodies at 22 DAP (days after pollination) showed that the protein bodies were morphologically similar to those of normal genotypes. However, the endosperm ofopaque-2 maize at 14 DAP contains tubular arrays within the rough endoplasmic reticulum. These tubular arrays are tightly associated with the developing protein bodies. Long strands of tubules, sometimes 10 m in length, are observed in the endosperm, and partially formed protein bodies often seem to be forming directly from these tubular arrays. No immunostaining is associated with this tubular material when any of the anti-zein antibodies are used.Abbreviations BSA bovine serum albumin - DAP days after pollination - IgG immunoglobulin G Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Expression of protein disulfide isomerase is elevated in the endosperm of the maize floury-2 mutant

A maize protein disulfide isomerase (PDI, EC 5.3.4.1) cDNA clone was isolated and characterized. The deduced amino acid sequence contains two regions characteristic of the active sites for PDI and a carboxyl-terminal endoplasmic reticulum (ER) retention sequence, Lys-Asp-Glu-Leu. Southern blot analysis indicated the maize PDI is encoded by a single gene that maps to the short arm of chromosome 4. When isolated from the cisternal and protein body ER, the PDI protein resolves into a fast and a slow form on SDS-PAGE. During endosperm development, the PDI RNA level increases between 10 and 14 days after pollination. In floury-2 (fl2) endosperm, which contains an abnormally processed -zein protein, PDI expression is significantly increased, and the level of PDI protein and RNA is positively correlated with the dosage of fl2 alleles. The increase of PDI in fl2 occurs mainly in the cisternal ER fraction, whereas the most dramatic increase of binding protein (BiP) is in the protein body ER. We propose that the induction of PDI in the fl2 mutant reflects its role as a molecular chaperone, and that PDI functions in concert with BiP at different stages of zein processing and assembly into protein bodies.     

Cotton embryogenesis: The pollen cytoplasm

Summary The ultrastructure and composition of cotton (Gossypium hirsutum) pollen, exclusive of the wall, was examined immediately before and after germination. The pollen grain before germination consists of two parts: the outer layer and a central core. The outer layer contains large numbers of mitochondria and dictyosomes as well as endoplasmic reticulum (ER). The core contains units made of spherical pockets of ER which are lined with lipid droplets and filled with small vesicles; the ER is rich in protein and may contain carbohydrate while the vesicles are filled with carbohydrate. Starch-containing plastids are also present in the core as are small vacuoles. The cytoplasm of the pore regions contains many 0.5 spherical bodies containing carbohydrate. After germination the ER pockets open and the lipid droplets and small vesicles mix with the other portions of the cytoplasm. With germination the pore region becomes filled with mitochondria and small vesicles. The vegetative nucleus is large, extremely dense and contains invaginations filled with coils of ER. A greatly reduced nucleolus is present in the generative cell which is surrounded by a carbohydrate wall. The cytoplasm of the generative cell is dense and contains many ribosomes, a few dictyosomes and mitochondria, many vesicles of several sizes, and some ER. No plastids were identified. The generative nucleus is also dense with masses of DNA clumped near the nuclear membrane. An unusual tubular structure of unknown origin or function was observed in the generative cell.     

The endomembrane system of differentiating carposporangia in the red algaChondria tenuissima: Occurrence and participation in secretion of polysaccharidic and proteinaceous substances

Summary The endomembrane system during carposporogenesis inChondria tenuissima was studied using electron microscopy and histochemistry. Profiles of the nucleus are convoluted, resulting in a highly increased surface area. Stacked cisternae are found within the peripheral part of the nucleus. Vesicles, tubules and membrane bound fibrillar bodies occur within the nucleoplasm. The endoplasmic reticulum surrounds the nuclear envelope.The endoplasmic reticulum and the Golgi apparatus, together with small transition vesicles, represent a functional unit. They form two different secretory substances during carposporogenesis. In young stages, carbohydrates are produced by normal dictyosomes within large, normal exocytotic Golgi vesicles. They do not react positively with PAS or Thiéry method and are believed to represent cell wall material. In later stages, the central area of the Golgi cisternae becomes filled with electron dense material. The individual cisternae are transformed into cored vesicles at the trans-face of the dictyosomes. The dense core of the vesicles is proteinaceous and stains with coomassie brilliant blue R. The peripheral fibrillar material is polysaccharidic and reacts positively using the Thiéry method. The contents of the cored vesicles are believed to participate in carpospore attachment. The ER gives rise to cytolysosomes in which starch grains are sequestrated and digested. Mucilaginous sacs seem to be similarly formed.     

Studies of pollen maturation in cotton: the storage reserve accumulation phase

Summary This study follows the maturation of the pollen grain of cotton (Gossypium hirsutum L.), particularly the development of the vegetative cytoplasm and the various storage products formed. CTEM, HVEM, stereoscopy, and cyto-histochemistry were used to examine the events occurring during the 9 days before anthesis. Starch began to accumulate in plastids at anthesis minus 9 days and reached a peak concentration shortly before anthesis; lipid deposition followed a similar pattern, but started at 6 days before anthesis. Lipid bodies were always seen closely oppressed to the endoplasmic reticulum (ER). Dictyosomes appear active during the entire 9 days; first producing vesicles involved in the formation of the intine and, later, producing vesicles stored in the pollen grain. The dictyosome vesicles appear to contain polysaccharides and concentrate in layers around the lipid bodies. Ribosomes increase in number from 6 days before anthesis and are particularly numerous in the mature pollen. From anthesis minus 6 days until anthesis, the ER cisternae become increasingly inflated and, in the hours immediately before pollen release, form pockets filled with lipid bodies and dictysosome vesicles. The mature pollen has a core region filled with ER pockets and a peripheral cytoplasm in which such pockets are generally lacking.This research was supported in part by NSF Grant BMS575-22-23 and Grant N.RR-00592 from the Division of Research Resources, National Institutes of Health     

荞麦糊粉层和子叶中贮藏蛋白质积累过程的超微结构

应用透射电镜技术对荞麦（Fagopyrum esculentum)子叶和糊粉层细胞中贮藏蛋白质的积累过程进行了研究。荞麦开花后15天,胚乳最外细胞的液泡中开始积累蛋白质。开花后25天,最外层胚乳细胞中积累较多的糊粉粒（直径1－2μm)形成糊粉层。开花后20天,子叶细胞中蛋白质开始在液泡和细胞质中积累,同时液泡通过膜的向内生长和缢裂两种方式形成体积较小的液泡。开花后25天,成熟的子叶细胞中含有丰富的蛋白质,贮藏蛋白质主要积累在液泡中形成体积较大的蛋白质贮藏液泡（PSVs,protein storage vacuoles,直径1－3μm）。在荞麦子叶积累蛋白质的各个阶段,细胞质中都有一些来源于高尔基体,含蛋白质的电子不透明小泡（直径0.1-0.7μm)存在,观察到有些小泡正进入液泡,推断这种来自高尔基体膜囊的小泡不仅将蛋白质运输到液泡形成PSVs的作用,也可能是荞麦成熟子叶积累贮藏蛋白质的一种结构。     

Immunogold localization of chymotrypsin inhibitor-2, a lysine-rich protein,in developing barley endosperm

Chymotrypsin inhibitor-2, a lysine-rich protein in the barley endosperm, has been localized at the ultrastructural level by immunocytochemistry in developing barley endosperm cells 14 days post anthesis. The protein is deposited in the protein bodies. Two morphologically distinct types of protein bodies, small spherical and large irregularly shaped, are present. Golgi-apparatus-derived vesicles whose content is labelled by chymotrypsin inhibitor-2 antibody-gold particles are observed at the Golgi complex and around the vacuoles. These observations indicate that the transport of the protein to the site of deposition is mediated by the Golgi apparatus.Abbreviations CI chymotrypsin inhibitor - DPA days post anthesis - ER endoplasmic reticulum The authors wish to thank Dr. V.R. Franceschi (Department of Botany, Washington State University, Pullman, USA) for many helpful discussions and advice during the work, and the staff at the Electron Microscope Center at Washington State University for technical assistance.