The cell wall of the chlamydomonad flagellate,Gloeomonas kupfferi (Volvocales,Chlorophyta)

Summary The large unicellular flagellate,Gloeomonas kupfferi, has recently been used as an important tool in chlamydomonad cell biology research, especially in studies dealing with the structure and function of the endomembrane system. However, little is known about the main secretory product, the cell wall. This study presents structural, chemical and immunological information about this wall. This 850–900 nm thick matrix is highly elaborate and consists of three distinct layers: an inner stratum (325 nm thick) consisting of tightly interwoven fibers, a medial crystalline layer consisting of 22–23 nm subunits and an outer wall layer (500 nm thick) of outwardlyradiating fibrils. Rapid freeze-deep etch analysis reveals that the 35–40 nm fibers of the outer layer form a quasi-lattice of 160 nm subunits. The outer wall can be removed from whole pellets using the chelator, CDTA. The medial wall complex can be solubilized by perchlorate. SDS-gel electrophoresis reveals that the perchlorate soluble-material consists of five high molecular weight glycoproteins and five major low molecular weight glycoproteins. The electrophoretic profile is roughly similar to that ofChlamydomonas reinhardtii. Antibodies were successfully raised against the outer wall component and were shown to label the outer wall layer.     

The endomembrane system and mechanism of membrane flow in the green alga,Gloeomonas kupfferi (Volvocales,Chlorophyta) I. An ultrastructural analysis

Summary The endomembrane system of the chlamydomonad flagellate,Gloeomonas kupfferi (Chlorophyta), is complex. It consists of a proliferating ER network, a perinuclear complex of 14–18 dictyosomes and 8–12 vacuoles and an anterior contractile vacuole complex. The ER network extends from the nuclear envelope outwards, ensheafhs a dictyosome, extends out through a lobe of the chloroplast and terminates in the thin zone of peripheral cytoplasm between the chloroplast and plasmamembrane. The individual dictyosome is polar with distinct cis- and trans-faces. The cis-face is closely associated with transition vesicles emerging from the adjacent ER. Large vesicles emerge from peripheral swellings of terminal cisternae. The dictyosome-associated ER is connected to the peripheral vacuolar system. During cell division and cytokinesis, changes in the endomembrane system occur. Dictyosomes divide and quickly separate to form perinuclear complexes around the daughter nuclei. Each dictyosome undergoes morphological changes during this wall precursor-producing stage. ER lines the furrow zone and is closely associated with phycoplast microtubules. A discussion of the endomembrane system in membrane flow mechanics is provided.Abbreviations ER endoplasmic reticulum - OsFeCN Osmium ferricyanide     

An autolysin dissolves the inner cell wall layer of the algaPediastrum (Hydrodictyaceae)

Summary An autolysin produced by young colonies ofPediastrum frees them from the vesicle in which they are formed within 12 hours of release of zoospores from the parent cell. The polysaccharide vesicle is derived from the inner wall layer of the parent cell. Refrigeration delays vesicle disintegration; boiling stops it completely. A purified, lyophilized extract of the vesicle fluid added to boiled vesicled colonies removes the vesicle in 2 hours with the release of reducing sugars and polysaccharides.Biogel P2 and P10 chromatography of the products following incubation of the enzyme preparation and wall showed no more than 1% oligosaccharides; the remaining carbohydrates had a molecular weight of several thousand daltons. Analyses of isolated vesicle wall material (70–85% of the dry weight) showed mannose accounting for approximately 50% of the dry weight, with none of the other neutral sugars present (fucose, xylose, galactose and glucose) representing more than 3%. Uronic acids account for 20–25% of the wall weight, and proteins less than 2%. Pediastrum colonies are thus freed from the vesicles in which they are formed by the action of an autolysin they produce. The autolysin acts on the vesicle wall material to generate reducing sugars and cause it to disintegrate into its constituent polysaccharides.     

The endomembrane system and mechanism of membrane flow in the green alga,Gloeomonas kupfferi (Volvocales,Chlorophyta) II. A cytochemical analysis

Summary Cytochemical analysis of the endomembrane system of the chlamydomonad flagellate,Gloeomonas kupfferi (Chlorophyta), reveals distinct compartmentalization. Phosphatase localization shows that: IDPase is located throughout all cisternae of the dictyosome and vesicles associated with the contractile vacuole. Other alkaline phosphatases like TPPase, ATPase and ITPase were localized within the trans-face cisternae and vesicles of the contractile vacuole. IMPase was localized at the plasmamembrane and not within the endomembrane system. Acid phosphatases, incl. CMPase, NADPase and -glycerophosphatase, were localized in vesicles emerging from the central terminus of the trans-face of the dictyosome and in the peripheral vacuolar network. Silver proteinate labeling was noted in the dictyosome, contractile vacuole and on the anterior plasmamembrane. A summary of endomembrane compartmentalization and a putative interpretation of membrane flow and economy are presented.Abbreviations ER endoplasmic reticulum - IDPase inosine 5-diphosphatase - ITPase inosine 5-triphosphatase - ATPase adenosine 5-triphosphatase - TPPase thiamine pyrophosphatase - CMPase cytidine 5-monophosphatase - NADPase -nicotinamide adenine diphosphatase - AcPase acid phosphatase     

Changes in the secretory activity of the Golgi apparatus during the cell cycle in root tips of maize (Zea Mays L.)

Epidermal cells of maize roots were studied to determine the distribution of Golgi apparatus-derived secretory vesicles in various stages of cell division. The following conclusions were reached: 1) The pattern of Golgi apparatus secretion varies with the cell cycle. 2) Large numbers of secretory vesicles are incorporated into the cell plate. 3) Secretory vesicles from the Golgi apparatus are incorporated primarily in walls undergoing expansion. 4) Secretory vesicles are smaller during mitosis and the first part of cytokinesis than they are during interphase. 5) Secretory vesicles account for at least 12–23% of cell-plate plasma membrane and an estimated 25% of cell-plate volume.     

The change of pattern in microfibril arrangement on the inner surface of the cell wall of Closterium acerosum during cell growth

Closterium acerosum (Schrank) Ehrenberg cells cultured on cycles of 16 h light and 8 h dark, undergo cell division synchronously in the dark period. After cell division, the symmetry of the daughter semicells is restored by controlled expansion, the time required for this restoration, 3.5–4 h, being relatively constant. The restoration of the symmetry is achieved by highly oriented surface expansion occurring along the entire length of the new semicell. During early semicell expansion, for about 2.5 h, microfibrils are deposited parallel to one another and transversely to the cell axis on the inner surface of the new wall. Wall microtubules running parallel to the transversely oriented microfibrils are observed during this period. About 2.5 h after septum formation, preceding the cessation of cell elongation, bundles of 7–11 microfibrils running in various directions begin to overlay the parallel-arranged microfibrils already deposited. In the fully elongated cells, no wall microtubules are observed.     

Immunolocalization of the cell wall components inPinus densiflora pollen

Summary In order to compare cell wall formation in gymnosperm pollen with that in angiosperm pollen, the distribution of cell wall constituents in the pollen grain and pollen tube ofPinus densiflora was studied immunocytochemically with monoclonal antibodies JIM 5 (against non- or poorly esterified pectin), JIM 7 (against highly esterified pectin), JIM 13 (against arabinogalactan proteins, AGPs), and LM 2 (against AGPs containing glucuronic acid). In the pollen grain wall, only the outer layer of the intine was labeled with JIM 5 and weakly with JIM 7. The tube wall was scarcely labeled with JIM 5 and very weakly labeled with JIM 7. In contrast, the whole of both the intine and the tube wall was strongly labeled with JIM 13 and LM 2, and the generative-cell wall was also labeled only with LM 2. The hemicellulose B fraction, which is the main polysaccharide fraction from the pollen tube wall, reacted strongly with JIM 13 and especially LM 2, but not with antipectin antibodies. These results demonstrate that the wall constituents and their localization inP. densiflora pollen are considerably different from those reported in angiosperm pollen and suggest that the main components of the cell wall ofP. densiflora pollen are arabinogalactan and AGPs containing glucuronic acid.Abbreviations AGPs arabinogalactan proteins - ELISA enzymelinked immunosorbent assay - MAbs monoclonal antibodies     

Composition of the cell wall of Chlorella fusca

Isolated cell walls of mature Chlorella fusca consisted of about 80% carbohydrate, 7% protein, and 13% unidentified material. Mannose and glucose were present in a ratio of about 2.7:1 and accounted for most of the carbohydrate. Minor components were glucuronic acid, rhamnose, and traces of other sugars; galactose was absent. After treatment with 2 M trifluoroacetic acid or with 80% acetic acid/HNO₃ (10/1, v/v), a residue with a mannose/glucose ratio of 0.3:1 was obtained, probably representing a structural polysaccharide. An X-ray diffraction diagram of the walls showed one diffuse reflection at 0.44 nm and no reflections characteristic of cellulose. Walls from young cells contained about 51% carbohydrate, 12% protein, and 37% unidentified material. Mannose and glucose were also the main sugars; their absolute amounts per wall increased 6–7 fold during cell growth. Walls isolated with omission of a dodecylsulphate/mercaptoethanol/urea extraction step had a higher protein content and, with young walls, a significantly higher glucose and fucose content. These data and other published cell wall analyses show a wide variability in cell wall composition of the members of the genus Chlorella.Abbreviations GLC gas liquid chromatography - TFA trifluoroacetic acid     

The effects of brefeldin A upon the Golgi apparatus of the green algal flagellate, Gloeomonas kupfferi

The fungal-derived derivative, brefeldin A, was used to disruptthe Golgi apparatus (GA) of the green alga, Gloeomonas kupfferi.Upon short treatments (10 µg ml^–1 for 10 min orless), the Golgi bodies maintain their perinuclear positioning.However, the medial locus transforms from a tight stack of elongatecisternae to a network of swollen tubules. Upon longer treatments(60 min), swelling and vesiculation of cis face cisternae becomeapparent. Likewise, the edges of several trans face cisternaemay fuse with those of adjacent Golgi bodies leading to theformation of multiGolgi complexes. Key words: Brefeldin A, Golgi apparatus, Gloeomonas kupfferi     

Growth kinetics of the Golgi apparatus during the cell cycle in onion root meristems

In onion root meristems, the number of dictyosomes per cell shows a kinetics of growth strongly related to the cell cycle. During the interphase of steady-state proliferative cells, the volume density and numerical density of the Golgi apparatus decrease to reach minimum values in late-interphase cells, characterized by their greatest length. This pattern is also found in the total volume occupied by Golgi apparatus. Once in mitosis, the above-mentioned parameters begin to increase reaching maximum mean values in telophase. After the experimental uncoupling of chromosome and growth cycles by presynchronization with hydroxyurea, we found a similar behaviour pattern in the Golgi apparatus: decreasing values during interphase and a triggering of Golgi-apparatus growth in prophase independently of the bigger cell sizes reached in mitosis as an effect of pretreatment with hydroxyurea. These results indicate a cyclic kinetics of this subcellular component in higher-plant meristems, coupled with early mitotic events.     

Some observations on cell wall structure and taxonomy ofPhymatodocis nordstedtiana (Conjugatophyceae,Chlorophyta)

Filaments ofPhymatodocis nordstedtiana Wolle were isolated from a sample of a Texan lake. Cultures were established and examined by light and scanning electron (SEM) as well as transmission electron microscopy (TEM). It is shown that the pores apparent on light microscopical examination are not of the cosmaroid type as expected. TEM examination disclosed that they are similar to those found in the generaClosterium Ralfs andPenium Bréb. Furthermore, it could be demonstrated by light and SEM microscopy that the primary cell wall is shed during cell division. The remaining secondary cell wall of the mature cell consists of interwoven bands of parallel microfibrils. A conspicuous overlap of the semicell walls clearly denotes the isthmus region. The significance of these deviations unusual for desmids is discussed. Suggestions are made that the taxonomic position ofPh. nordstedtiana should be re-evaluated.     

Immunogold localization of the cell-wall-matrix polysaccharides rhamnogalacturonan I and xyloglucan during cell expansion and cytokinesis inTrifolium pratense L.; implication for secretory pathways

We have localized two cell-wall-matrix polysaccharides, the main pectic polysaccharide, rhamnogalacturonan I (RG-I), and the hemicellulose, xyloglucan (XG), in root-tip and leaf tissues of red clover (Trifolium pratense L.) using immunoelectron microscopy. Our micrographs show that in both leaf and root tissues RG-I is restricted to the middle lamella, with 80–90% of the label associated with the expanded regions of the middle lamella at the corner junctions between cells. Xyloglucan, however, is nearly exclusively located in the cellulose-microfibril-containing region of the cell wall. Thus, these cell-wall-matrix polysaccharides are present in distinct and complementary regions of the cell wall. Our results further show that during cell expansion both RG-I and XG are present within Golgi cisternae and vesicles, thus confirming that the Golgi apparatus is the main site of synthesis of the non-cellulosic cell-wall polysaccharides. No label is seen over the endoplasmic reticulum, indicating that synthesis of these complex polysaccharides is restricted to the Golgi. The distribution of RG-I and XG in root-tip cells undergoing cell division was also examined, and it was found that while XG is present in the Golgi stacks and cell plate during cytokinesis, RG-I is virtually absent from the forming cell plate.Abbreviations ER endoplasmic reticulum - RG-I rhamnogalacturonan I - XG xyloglucan     

Separation of membranes by flotation centrifugation for in vitro synthesis of plant cell wall polysaccharides

Summary Membranes from etiolated maize seedlings were isolated using sucrose gradients for in vitro studies of polysaccharide synthesis. Following downward centrifugation, flotation centrifugation improved the purity of membrane fractions, in particular the Golgi apparatus. Based on naphthylphthalamic acid binding to plasma membrane and inosine-5-diphosphatase activity in Golgi apparatus, flotation centrifugation removed about 70% of the plasma membrane which cosedimented with the Golgi apparatus in downward centrifugation. The addition of chelators during flotation centrifugation allowed separation of the Golgi apparatus from endoplasmic reticulum, as indicated by NADH cytochromec reductase activity. Glucan and xylan synthase activities were measured as the radioactivity incorporated from either UDP-¹⁴C-glucose or UDP-¹⁴C-xylose into 80% ethanol insoluble materials. Glucan synthase activity at a substrate concentration of 1 mM UDP-glucose without CaCl₂ was greatest in fractions enriched in Golgi apparatus, but in the presence of 3 mM CaCl₂ the activity was greatest in fractions enriched in plasma membrane. Glucan synthase activity at a substrate concentration of 10M UDP-glucose in the presence of 3 mM MnCl₂ was greatest in fractions enriched in plasma membrane, but was also high in fractions enriched in Golgi apparatus. Xylan synthase activity, at a substrate concentration of 1 M UDP-xylose in the presence of 3 mM MnCl₂, was greatest in fractions enriched in Golgi apparatus. To further characterize these synthase reactions, the glycosyl linkages of the products formed were analyzed with a gas chromatograph coupled to a radiogas proportional counter. With the substrate, UDP-¹⁴C-glucose, and fractions enriched in Golgi apparatus, both (13)- and (14)-radioactive glucosyl linkages were formed, whereas the main linkage formed by fractions enriched in plasma membrane was (13)-glucosyl. With the substrate, UDP-¹⁴C-xylose, mostly (14)-xylosyl and some terminal-xylosyl linkages were formed by fractions enriched in Golgi apparatus. Only xylan synthase activity copurified with Golgi apparatus and, because plasma membrane lacked this activity, xylan synthase may be used as a reasonable indicator of Golgi apparatus.Abbreviations ATP adenosine-5-triphosphate - CR crude fraction from downward centrifugation - FL purified fraction from flotation centrifugation - GC gas chromatography - GC-RPC gas chromatography-radiogas proportional counting - IDP inosine-5-disphosphate - NPA naphthylphthalamic acid - UDP uridine-5-diphosphate - TEM transmission electron microscopy     

The cell wall of Chlamydomonas reinhardtii gametes: Composition,structure and autolysin-mediated shedding and dissolution

The cell walls of Chlamydomonas gametes are multilayered structures supported on frameworks of polypeptides extending from the plasma membrane. The wall-polypeptide catalogue reported by Monk et al. (1983, Planta 158, 517–533) and extended by U.W. Goodenough et al. (1986, J. Cell Biol. 103, 405–417) was re-evaluated by comparative analysis of mechanically isolated cell walls purified from several strains. The extracellular locus of wall polypeptides was verified by in vivo iodogen-catalysed iodination and by autolysin-mediated elimination of the bulk of these polypeptides from the cell surface. Three (w15, w16, w17) and possibly four (w14) polypeptides were located to the most exterior aspect of the wall because of their susceptibility to Enzymobeadcatalysed iodination and their retention by a cell-wall-less mutant. The composition of shed walls stabilised with ethylenediaminetetraacetic acid during natural mating and kinetic analysis of the dissolution of walls purified from a bald-2 mutant demonstrated the rapid and specific destruction of polypeptide w3. Differential solubilisation of wall polypeptides occurred after loss of w3. Wall dissolution, characterised by the generation of fishbone structures from the W2 layer, gave as many as four additional polypeptides. Charged detergents and sodium perchlorate extracted a comparable range of polypeptides at room temperature from mechanically isolated walls, i.e. components of the W4–W6 layers, hot sodium dodecyl sulphate solubilised framework polypeptides, while reducing agent was required to solubilise the W2 layer. A model of wall structure is presented.Abbreviations DTE dithioerythritol - EDTA ethylenediaminetetraacetic acid - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

N-Glycyl-glucosamine: A novel constituent in the cell wall of Halococcus morrhuae

The dinitrophenyl-derivative of N-glycylglucosamine was isolated from partially hydrolyzed dinitrophenylated cell walls of Halococcus morrhuae CCM 859. To increase the yield of amino-terminal glycine residues, halococcal cell walls were treated with alkali or acid prior to dinitrophenylation. Authentic N-glycyl-glucosamine was used as a reference substance. A substitution of the amino group of glucosamine by an amino acid has so far not been found in any other wall of a pro- or eucaryotic cell. Since only 5% of the glycine residues reveal an unsubstituted carboxyl group within intact cell walls, glycine may play a role in connecting glycan strands through peptidic linkages between the amino group of glucosamine and the carboxyl group of an uronic acid or gulosaminuronic acid.Abbreviations DNP dinitrophenyl     

Changes in the activity of the Golgi apparatus during the cell cycle in antheridial filaments ofChara vulgaris L.

Summary The number of dictyosomes found in one central cell section in antheridial filaments ofChara vulgaris increases proportionally to the cell length during interphase. The activity of Golgi apparatus was expressed by a number of Golgi vesicles surrounding a single dictyosome. These vesicles are most numerous during mitosis and cytokinesis,i.e., prior to and during cell plate formation. In the middle and late S phase the number of Golgi vesicles decreases by about 25%; subsequently, during the early and middle G₂, it increases again. At the end of the G₂ phase, Golgi vesicles are the scarcest.The increase in the number of Golgi vesicles during the G₂ phase coincides with the period of intense cellular elongation, and, thus, it is probably related to the enhanced synthesis of cell wall components.Coated vesicles are most numerous in prophase, metaphase, and early telophase, and during interphase in both late S and G₂ phase. It was found that the number of coated vesicles is proportional to the degree of condensation of nuclear chromatin.This work was supported by the Polish Academy of Sciences within the project 09.7.3.1.4.     

Ultrastructural and immunochemical features of the cell wall sac formed in mulberry (Morus alba) idioblasts

A peculiar inward growth, named a “cell wall sac”, formed in mulberry (Morus alba) idioblasts, is a subcellular site for production of calcium carbonate crystals. On the basis of ultrastructural observations, a fully expanded cell wall sac could be divided into two parts—an amorphous complex consisting of multi-layered compartments with multiple fibers originating from the innermost cell wall layer, and a peripheral plain matrix with fiber aggregates. Immunofluorescent localization showed that low and highly esterified pectin epitopes were detected at the early stages of development of the cell wall sac, followed by complete disappearance from the both parts of fully enlarged mature sac. In contrast, the xyloglucan epitope remained in the compartment complex; this was supported by the observation that the xyloglucan epitope labeled with immuno-gold particles is found on fibers in the complex part.     

Electron microscopy of the cell wall complex ofMethylomonas albus

In surface view, the cell wall complex ofMethylomonas albus possesses a hexagonal pattern of ridges. Thin sections reveal a continuous layer of goblet-shaped elements attached to the outermost surface of the lipopolysaccharide membrane. A possible interpretation of the cell wall complex ofM. albus, based on the fine-structural data is presented.     

Effects of colchicine on cell shape and on microfibril arrangement in the cell wall of Closterium acerosum

Cell morphogenesis in Closterium acerosum (Schrank) Ehrenberg was greatly influenced by colchicine. Addition of colchicine to the medium led to production of tadpole-shaped cells, by decreasing the length and increasing the thickness of the new semicells. Transversely oriented wall microtubules and microfibrils, characteristic of normally elongating semicells, were not observed in colchicine-treated semicells, randomly oriented microfibrils being present instead. About 3.5 h after septum formation, the randomly oriented microfibrils began to be overlaid by bundles of microfibrils as seen in normal semicells at the later stage of elongation. When colchicine treatment was terminated 1 h after septum formation, cell elongation was partially restored and microfibrils were deposited parallel to each other and transversely to the cell axis, indicating that the effect of colchicine on microfibril arrangement in growing semicells is reversible.     

A 41 kDa protein isolated from maize mesocotyl cell walls immunolocalizes to plasmodesmata

Summary Plasmodesmata, dynamic pore structures that traverse plant cell walls, function in cytoplasmic transport between contiguous cells. Cell walls containing embedded plasmodesmata were isolated from mesocotyls of etiolated maize seedlings. Proteins associated with the isolated walls were separated by SDS-PAGE and antibodies were generated against a 41 kDa protein, one of several associated with this wall fraction. Immunoblot analysis showed that the 41 kDa polypeptide was also associated with other subcellular fractions obtained following tissue homogenization and differential centrifugation. The wall associated 41 kDa protein is apparently a peripheral membrane protein since it could be extracted by high salt and high pH. Silver-enhanced immunogold light microscopy showed that the 41 kDa protein was associated with the cell walls of cells both in the stele and cortex. The immunolabeling pattern was transwall and punctate. Electron microscopic immuno-gold labeling localized the polypeptide to plasmodesmata and to electron dense cytoplasmic structures that are apparently Golgi membranes. The significance of the presence of this protein in the Golgi is discussed.Abbreviations ER endoplasmic reticulum