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.     

Subunits of forming pollen exine and Ubisch bodies as seen in freeze substitutedLedebouria socialis Roth (Hyacinthaceae)

Summary High-pressure freezing/freeze substitution/TEM was employed to investigate anthers of the monocotyledonous angiospermLedebouria socialis Roth (Hyacinthaceae) during early tetrad stage. The initials of the outer sporopollenous pollen wall stratum (=sexine) and of the homologous tapetal products (=Ubisch bodies) are composed of highly regular subunits: clustered globules with a constant diameter of approximately 28 nm. The clusters develop within diffuse accumulations of electron-dense material. This process, interpreted as sporopollenin polymerization, does not necessarily depend on the presence of membrane-bound enzymes. Immunogold labeling with JIM 5 and JIM 7 antibodies revealed that the primexine as well as the dissolving tapetal cell walls, the sites of sexine and Ubisch body formation, respectively, contain un-esterified and methyl-esterified pectins.Abbreviations E-PTA ethanolic phosphotungstic acid - PA periodic acid - UA/Pb uranyl acetate/lead     

Ultrastructure of the pea cotyledon Golgi apparatus: Origin of dense vesicles and the action of brefeldin A

Summary We have followed the action of brefeldin A (BFA) on the Golgi apparatus of developing pea cotyledons, the cells of which are actively engaged in the synthesis and deposition of storage proteins. The Golgi apparatus of normal cells is characterized by the presence of three different types of vesicle: smooth-surfaced secretory vesicles, dense vesicles which carry the storage proteins, and clathrin-coated vesicles (CCV). The dense vesicles originate at the cis cisternae and undergo a maturation as they pass through the Golgi stack, presumably as a result of cisternal progression. CCV bud off from dense and smooth vesicles, which may be attached to one another, at the trans pole of the Golgi apparatus. BFA eliminates the CCV and leads, initially, to an increase in the number and length of the cisternae. Dense vesicles are still to be seen, and many show an increase in diameter. Longer BFA treatments result in a trans-driven vesiculation and an accumulation of vesicles within the vicinity of single cisternae. The vesicles were sometimes seen to be connected to one another via a network of tubules. As judged by immunocytochemistry with gold-coupled legumin and vicilin antisera, some of the dilated vesicles originate directly from dense vesicles by swelling whereas others probably arise by dilation of Golgi cisternae since they possess a layer of flocculent storage proteins at their periphery. By contrast the centre of the dilated vesicles labels positively with antibodies against complex glycans, indicating that the ability to segregate storage proteins from cell wall or lytic vacuole glycoproteins is lost during extended BFA treatment. The effects of BFA are reversible when cotyledons are further incubated on Gamborg's medium for 5 h without the inhibitor.Dedicated to Professor R. Kollmann on the occasion of his 65th birthday.     

Pectin and the role of the physical properties of the cell wall in pollen tube growth of<Emphasis Type="Italic"> Solanum chacoense</Emphasis>

The cell wall is one of the structural key players regulating pollen tube growth, since plant cell expansion depends on an interplay between intracellular driving forces and the controlled yielding of the cell wall. Pectin is the main cell wall component at the growing pollen tube apex. We therefore assessed its role in pollen tube growth and cytomechanics using the enzymes pectinase and pectin methyl esterase (PME). Pectinase activity was able to stimulate pollen germination and tube growth at moderate concentrations whereas higher concentrations caused apical swelling or bursting in Solanum chacoense Bitt. pollen tubes. This is consistent with a modification of the physical properties of the cell wall affecting its extensibility and thus the growth rate, as well as its capacity to withstand turgor. To prove that the enzyme-induced effects were due to the altered cell wall mechanics, we subjected pollen tubes to micro-indentation experiments. We observed that cellular stiffness was reduced and visco-elasticity increased in the presence of pectinase. These are the first mechanical data that confirm the influence of the amount of pectins in the pollen tube cell wall on the physical parameters characterizing overall cellular architecture. Cytomechanical data were also obtained to analyze the role of the degree of pectin methyl-esterification, which is known to exhibit a gradient along the pollen tube axis. This feature has frequently been suggested to result in a gradient of the physical properties characterizing the cell wall and our data provide, for the first time, mechanical support for this concept. The gradient in cell wall composition from apical esterified to distal de-esterified pectins seems to be correlated with an increase in the degree of cell wall rigidity and a decrease of visco-elasticity. Our mechanical approach provides new insights concerning the mechanics of pollen tube growth and the architecture of living plant cells.     

Thin section and freeze fracture studies of the hypophyseal proximal pars distalis in a teleost (Rhamdia hilarii Val.) during different stages of the reproductive cycle

Summary Chromophilic cells in the proximal pars distalis of the adenohypophysis of Rhamdia hilarii were studied in thin section and freeze fracture preparations. The gonadotropic cells (GTH-cells) exhibit a diversity of form, the frequency of which can be related to stages (maturation, mature and spent) in the sexual cycle. GTH-cells showing a cytoplasm filled with electron dense polymorphic secretory granules and small rough endoplasmic reticulum (RER) vesicles, have been termed non-vacuolated. During the mature gonadal stage, such cells become increasingly vacuolated. The small RER vesicles become dilated and/or fuse, forming a single enormous cisternum (4–11 m diameter), the contents of which show direct contact with the inner nuclear membrane. These morphological aspects support the idea that Rhamdia hilarii possesses only one GTH-cell type. Evidence from freeze fracture replicas suggests that membrane-associated events precursory to exocytosis take place in regions where the cell and secretory granule membranes are in close apposition. Thin section analysis of secretory granule formation revealed their derivation from the dilated extremities of the inner Golgi saccule which appears to resemble the rigid lamella described in other cells. After detachment of the inner saccule, the immature secretory granules appear to enlarge by microvesicular transport. Freeze fracture and ultrastructural data on the morphology of the cells that presumably synthetise growth hormone are also presented.This work was aided by the Fundação de Amparo à Pesquisa do Estado de São Paulo (75/1282)     

Recent developments in the cell and molecular biology of root hairs

Recent results in root hair research show that these tip-growing cells are useful models in plant cell biology research. The review covers a range of topics, but there is particular emphasis on the use of mutants in molecular (genetic) analysis.     

Erv41p and Erv46p: new components of COPII vesicles involved in transport between the ER and Golgi complex

Proteins contained on purified COPII vesicles were analyzed by matrix-assisted laser desorption ionization mass spectrometry combined with database searching. We identified four known vesicle proteins (Erv14p, Bet1p, Emp24p, and Erv25p) and an additional nine species (Yip3p, Rer1p, Erp1p, Erp2p, Erv29p, Yif1p, Erv41p, Erv46p, and Emp47p) that had not been localized to ER vesicles. Using antibodies, we demonstrate that these proteins are selectively and efficiently packaged into COPII vesicles. Three of the newly identified vesicle proteins (Erv29p, Erv41p, and Erv46p) represent uncharacterized integral membrane proteins that are conserved across species. Erv41p and Erv46p were further characterized. These proteins colocalized to ER and Golgi membranes and exist in a detergent-soluble complex that was isolated by immunoprecipitation. Yeast strains lacking Erv41p and/or Erv46p are viable but display cold sensitivity. The expression levels of Erv41p and Erv46p are interdependent such that Erv46p was reduced in an erv41Delta strain, and Erv41p was not detected in an erv46Delta strain. When the erv41Delta or ev46Delta alleles were combined with other mutations in the early secretory pathway, altered growth phenotypes were observed in some of the double mutant strains. A cell-free assay that reproduces transport between the ER and Golgi indicates that deletion of the Erv41p-Erv46p complex influences the membrane fusion stage of transport.     

Golgi apparatus activity and membrane flow during scale biogenesis in the green flagellateScherffelia dubia (Prasinophyceae). II: Cell wall secretion and assembly

Summary Secretion of the cell wall (theca) in the scaly green flagellateScherffelia dubia (Prasinophyceae) has been examined by electron microscopy during cytokinesis. The bi-laminate wall forms by the extracellular amalgamation of two layers of scales produced in the Golgi apparatus (GA). Each mature GA cisterna contains ca. 12,000 scales of two distinct varieties arranged in two layers on the cisternal membrane. GA cisternae undergo turnover and one scale containing cisterna matures from thetransface of each dictyosome every 3–4 minutes. Cisternae then fuse with the plasma membrane at the anterior end of the cell releasing the scales onto the cell surface. The two layers of wall scales integrate on the cell surface in a time-dependent self-assembly process. The first scales deposited commence assembly at the cell posterior and the wall develops anteriorly by edge growth. The daughter cell wall is composed of ca. 1.2 million scales deposited in about 3 hours. Calculations of net membrane flow strongly indicate extensive endocytosis during wall deposition.     

Changes in composition of the outer epidermal cell wall of pea stems during auxin-induced growth

The gross composition of the outer epidermal cell wall from third internodes of Pisum sativum L. cv. Alaska grown in dim red light, and the effect of auxin on that composition, was investigated using interference microscopy. Pea outer epidermal walls contain as much cellulose as typical secondary walls, but the proportion of pectin to hemicellulose resembles that found in primary walls. The pectin and hemicellulose fractions from epidermal peels, which are enriched for outer epidermal wall but contain internal tissue as well, are composed of a much higher percentage of glucose and glucose-related sugars than has been found previously for pea primary walls, similar to non-cellulosic carbohydrate fractions of secondary walls. The epidermal outer wall thus has a composition rather like that of secondary walls, while still being capable of elongation. Auxin induces a massive breakdown of hemicellulose in the outer epidermal wall; nearly half the hemicellulose present is lost during 4 h of growth in the absence of exogenous sugar. The percentage breakdown is much greater than has been seen previously for whole pea stems. It has been proposed that a breakdown of xyloglucan could be the basis for the mechanical loosening of the outer wall. This study provides the first evidence that such a breakdown could be occurring in the outer wall.M.S. Bret-Harte would like to thank Dr. Peter M. Ray, of Stanford University, for helpful discussions and for technical and editorial assistance, Dr. Winslow R. Briggs, of the Camegie Institude of Washington, for the use of experimental facilities and for helpful discussions, Dr. Wendy K. Silk, of the University of California, Davis, for helpful discussions and financial support, Dr. Paul B. Green for financial support, and Drs. John M. Labavitch and L.C. Greve, of the University of California, Davis, for performing the -cellulose analysis on short notice, in response to a request by an anonymous reviewer. This work was supported by a National Science Foundation Graduate Fellowship to M.S. B.-H., National Science Foundation Grant DCB8801493 to Paul B. Green, and the generosity of Wendy K. Silk (Department of Land, Air, and Water Resources, University of California, Davis) during the final writing.     

Characterization of the Neurospora crassa DHN melanin biosynthetic pathway in developing ascospores and peridium cells

Neurospora crassa contains all four enzymes for the synthesis of DHN (dihydroxynaphthalene), the substrate for melanin formation. We show that the DHN melanin pathway functions during N. crassa female development to generate melanized peridium and ascospore cell walls. N. crassa contains one polyketide synthase (PER-1), two polyketide hydrolases (PKH-1 and PKH-2), two THN (tetrahydroxynaphthalene) reductases (PKR-1 and PKR-2), and one scytalone dehydratase (SCY-1). We show that the PER-1, PKH-1, PKR-1 and SCY-1 are required for ascospoer melanization. We also identified the laccase that functions in the conversion of DHN into melanin via a free radical oxidative polymerization reaction, and have named the gene lacm-1 (laccase for melanin formation-1). In maturing perithecia, we show that LACM-1 is localized to the peridium cell wall space while the DHN pathway enzymes are localized to intracellular vesicles. We present a model for melanin formation in which melanin is formed within the cell wall space and the cell wall structure is similar to “reinforced concrete” with the cell wall glucan, chitin, and glycoproteins encased within the melanin polymer. This arrangement provides for a very strong and resilient cell wall and protects the glucan/chitin/glycoprotein matrix from digestion from enzymes and damage from free radicals.     

Ultrastructure of cell wall and photosynthetic apparatus of the phycobilisome-less Synechocystis sp. strain BO 8402 and phycobilisome-containing derivative strain BO 9201

The ultrastructures of two closely related strains of a novel diazotrophic cyanobacterium, Synechocystis sp. BO 8402 and BO 9201, were examined using ultrathin sections and freeze-fracture electron microscopy. Cells of both strains were surrounded by an unusual thick peptidoglycan layer. Substructures in the layer indicated the presence of microplasmodesmata aligned perpendicular to the free cell surface and in the septum of dividing cells. Synechocystis sp. strain BO 8402 contained lobed, electronopaque, highly fluorescent inclusion bodies consisting of phycocyanin-linker complexes. The thylakoids lacked phycobilisomes and accommodated, in addition to randomly distributed exoplasmic freeze-fracture particles, patches of two-dimensionally ordered arrays of dimeric photosystem II particles in the exoplasmic fracture face. Determination of photosystem I and photosystem II suggested an increase of photosystem II in strain BO 8402. Strain BO 9201 performed phycobilisome-supported photosynthesis and showed rows of dimeric photosystem II particles in the exoplasmic fracture face. Corresponding particle-free grooves in the protoplasmic fracture face were lined by a class of large particles tentatively assigned as trimers of photosystem I. The different lateral organization of protein complexes in the thylakoid membranes and the fine structure of the cell wall are discussed with respect to absorption cross-section of photosynthesis and nitrogen fixation.Abbreviations EF Exoplasmic freeze-fracture face - P 700 Reaction centre chlorophyll of photosystem I - PF Protoplasmic freeze-fracture face - PS I Photosystem I - PS II Photosystem II     

In-vivo observations of a spherical aggregate of endoplasmic reticulum and of Golgi vesicles in the tip of fast-growing Chara rhizoids

In-vivo differential interference contrast microscopy was used to detect individual Golgi vesicles and a new structure in the tip of fast-growing rhizoids of Chara fragilis Desvaux. This structure is a spherical clear zone which is free of Golgi vesicles, has a diameter of 5 m and is positioned in the center of the apical Golgi-vesicle accumulation (Spitzenkörper). After glutaraldehyde fixation and osmium tetroxide-potassium ferricyanide staining of the rhizoid, followed by serial sectioning and three-dimensional reconstruction, the spherical zone shows a tight accumulation of anastomosing endoplasmic reticulum (ER) membranes. The ER membranes radiate from this aggregate towards the apical plasmalemma and to the membranes of the statolith compartments. Upon gravistimulation the ER aggregate changes its position according to the new growth direction, indicating its participation in growth determination. After treatment of the rhizoid with cytochalasin B or phalloidin the ER aggregate disappears and the statoliths sediment. It is concluded that the integrity of the ER aggregate is actin-dependent and that it is related to the polar organisation of the gravitropically growing cell tip.Abbreviations CB cytochalasin B - DIC differential interference contrast microscopy - DMSO dimethyl sulfoxide - ER endoplasmic reticulum     

Auxin stimulates both deposition and breakdown of material in the pea outer epidermal cell wall,as measured interferometrically

The effect of auxin on the mass per area in the outer epidermal walls of third internodes of Pisum sativum L. cv. Alaska grown in dim red light was investigated using interference microscopy, and rates of net deposition of wall material were calculated. Examination of these net rates under different growth conditions showed that there is no simple relationship between the deposition of mass and growth. Net deposition can be proportional to growth when sufficient substrate for wall synthesis is available, as in intact plants, and in segments treated with indole-3-acetic acid (IAA) plus glucose. Net deposition can cause thickening of the walls when growth is small, as in the case of segments kept without IAA in the presence or absence of glucose, or segments whose growth is inhibited with mannitol. When substrate is limited and growth is large, however, wall expansion can occur with no net deposition, or an actual net loss of wall material can even take place. Auxin appears to induce a breakdown in the walls of segments treated in the absence of glucose, although it promotes synthesis when glucose is present. It is likely that IAA always induces a breakdown of wall material, but that the breakdown is masked when substrate is available for synthesis. Our results indicate that pea epidermal cells have two different auxin-stimulated mechanisms, wall synthesis and wall breakdown, potentially available to loosen their outer epidermal walls to bring about cell enlargement, alternatives which could be employed to different extents depending on substrate conditions.Abbreviation IAA indole-3-acetic acid M.S. Bret-Harte would like to thank Drs. Peter M. Ray, Stanford University, Winslow R. Briggs, Carnegie Institute of Washington, Stanford, Calif. USA, and Wendy K. Silk, of the University of California Davis USA, for helpful discussions, Dr. Briggs and the Carnegie Institute of Washington for the use of experimental facilities, and Dr. Ray for editorial assistance. This work was supported by a National Science Foundation Graduate Fellowship to M.S.B.-H., a National Science Foundation Postdoctoral Fellowship to T.I.B., and National Science Foundation grant DCB8801493 to P.B.G.     

A close temporal and spatial correlation between cell growth,cell wall synthesis and the activity of enzymes of mannan synthesis in Acetabularia mediterranea

The synthesis of cell wall mannan and the activities of guanosine-diphosphate-mannose-pyrophosphorylase (EC2.7.7.13) and mannan synthetase were studied during the development of nucleate and enucleated cells of the alga Acetabularia mediterranea. The activities of both enzymes are relatively high as long as the cells grow and synthesize mannans. With termination of growth and mannan synthesis, the activities of both enzymes, but especially of mannan synthetase, drop to a low value. Furthermore, the activities of both enzymes are distributed in the cell along an apical-basal gradient. High activities are present in the apical regions of the cell where growth and mannan synthesis mainly occur, whereas in the basal region, growth, mannan synthesis and the activity of the two enzymes are slight. Since the in vitro activity of GDP-Man-pyr is at least 100 times higher than that of mannan synthetase, it was concluded that mannan synthetase activity is the limiting factor in mannan synthesis. This conclusion is supported by the determined pool sizes of Fru 6-P, Man 6-P, Man 1-P and GDP-Man during the development of the cells. The control of mannan synthesis and with it cell wall formation and growth through the regulation of mannan synthetase activity is discussed.Abbreviations DD dark-dark regime - Fru 6-P fructose-6-phosphate - GDP-Man guanosine-diphosphate-mannose - GDP-Manpyr GDP-diphosphate-mannose-pyrophosphorylase - GTP guanosine-triphosphate - LD light-dark regime - Man 1-P mannose-1-phosphate - Man 6-P mannose-6-phosphate - TCA trichloracetic acid     

Gibberellic acid promotes growth and cell wall synthesis in Avena internodes regardless of the orientation of cell expansion

Segments cut from the next-to-last (peduncular-1) internode of Avena sativa L. cv. Victory (oat) shoots elongate as much as 10-fold in response to gibberellic acid (GA₃). The objective of the present investigation was to differentiate the effects of GA₃ on growth from its effects on wall synthesis (measured gravimetrically and through the incorporation of [¹⁴C]-glucose) by using several cell wall synthesis inhibitors with widely varying mechanisms of action. Four compounds, viz. monensin, cycloheximide, lanthanum, and galactose. caused (1) relatively little inhibition of either cell wall synthesis or elongation in segments without GA₃, (2) roughly proportionate, dose-dependent inhibition of elongation and wall synthesis in GA₃-treated segments and (3) generally greater inhibition of GA₃-promoted uptake of radioactivity than of wall incorporation or elongation. Two other compounds, colchicine and 2,6-dichlorobenzonitrile (DCB). (1) inhibited GA₃-induced elongation considerably more than cell wall synthesis and (2) caused swelling (radial expansion). especially of GA₃-treated segments. DCB-treated internodal cells apparently compensated for inhibited cellulose synthesis by greater synthesis of matrix polysaccharide (beginning between 3 and 6 h). While normal cellulose synthesis was not required for short-term (up to 6 h) GA₃-induced elongation or for long-term hormone-promoted radial expansion, it was required for sustained GA₃-induced elongation. These results indicate that GA₃-promoted cell wall loosening (manifested as radial expansion) and cell wall synthesis in Avena internodes occur at least partially independently of any hormonal effect on the orientation of microtubules and microfibrils.     

Cell-wall synthesis and elongation growth in hypocotyls of Helianthus annuus L.

The relationship between growth and increase in cell-wall material (wall synthesis) was investigated in hypocotyls of sunflower seedlings (Helianthus annuus L.) that were either grown in the dark or irradiated with continuous white light (WL). The peripheral three to four cell layers comprised 30–50% of the entire wall material of the hypocotyl. The increase in wall material during growth in the dark and WL, respectively, was larger in the inner tissues than in the peripheral cell layers. The wall mass per length decreased continuously, indicating that wall thinning occurs during growth of the hypocotyl. When dark-grown seedlings were transfered to WL, a 70% inhibition of growth was observed, but the increase in wall mass was unaffected. Likewise, the composition of the cell walls (cellulose, hemicellulose, pectic substances) was not affected by WL irradiation. Upon transfer of dark-grown seedlings into WL a drastic increase in wall thickness and a concomitant decrease in cell-wall plasticity was measured. The results indicate that cell-wall synthesis and cell elongation are independent processes and that, as a result, WL irradiation of etiolated hypocotyls leads to a thickening and mechanical stiffening of the cell walls.     

The ultrastructure of the cell surface and plasma membrane of exponential and stationary phase cells of Schizosaccharomyces pombe,grown in different media

In order to study the ultrastructure of the cell surface and plasma membrane of Schizosaccharomyces pombe as a function of growth conditions we investigated exponential and stationary phase cells grown in rich and minimal medium.Electron microscopic preparation techniques based on rapid cryofixation (without cryoprotectants) were used. The intramembraneous aspects of the plasma membrane were described by freeze fracturing. For the first time the dynamic surface structures could be directly analyzed by freeze drying in the scanning electron microscope and in thin section of freeze substituted samples. This preparation techniques reveal hair-like structures on the surface of yeast cells. The hairs of cells grown in the rich medium are longer than those grown in the minimal medium. A mutant defective in the structure of a cell surface galactomannoprotein (acid phosphatase) reveals (under conditions of maximal acid phosphatase expression) a cell surface structure that differs from the wild type. It is likely that the hairs represent the peripheral galactomannan layer or part of it.On the membrane fracture faces the number, shape, distribution and state of aggregation of the intramembraneous particles are different between membranes of growing and non-growing cells and between cells grown under different physiological conditions. In the minimal medium corresponding periodical structures on the plasmic and exoplasmic fracture faces were observed, which clearly differ between exponential and stationary phase cells. The number, length and depth of plasma membrane invaginations increase as the cells go from the exponential phase to the stationary phase. Short and flattened invaginations are filled with thin periodic structures.     

Microtubule reorganization,cell wall synthesis and establishment of the axis of elongation in regenerating protoplasts of the algaMougeotia

Summary Microtubule reorganization and cell wall deposition have been monitored during the first 30 hours of regeneration of protoplasts of the filamentous green algaMougeotia, using immunofluorescence microscopy to detect microtubules, and the cell-wall stain Tinopal LPW to detect the orientation of cell wall microfibrils. In the cylindrical cells of the alga, cortical microtubules lie in an ordered array, transverse to the long axis of the cells. In newly formed protoplasts, cortical microtubules exhibit some localized order, but within 1 hour microtubules become disordered. However, within 3 to 4 hours, microtubules are reorganized into a highly ordered, symmetrical array centered on two cortical foci. Cell wall synthesis is first detected during early microtubule reorganization. Oriented cell wall microfibrils, co-aligned with the microtubule array, appear subsequent to microtubule reorganization but before cell elongation begins. Most cells elongate in the period between 20 to 30 hours. Elongation is preceded by the aggregation of microtubules into a band intersecting both foci, and transverse to the incipient axis of elongation. The foci subsequently disappear, the microtubule band widens, and microfibrils are deposited in a band which is co-aligned with the band of microtubules. It is proposed that this band of microfibrils restricts lateral expansion of the cells and promotes elongation. Throughout the entire regeneration process inMougeotia, changes in microtubule organization precede and are paralleled by changes in cell wall organization. Protoplast regeneration inMougeotia is therefore a highly ordered process in which the orientation of the rapidly reorganized array of cortical microtubules establishes the future axis of elongation.