PLASMODESMATA AND AN ASSOCIATED CELL WALL COMPONENT IN BARLEY ALEURONE TISSUE

A unique cell wall component has been observed in the aleurone layer of barley (Hordeum vulgare L. cv. Himalaya). This wall component has been shown to be localized adjacent to the plasmalemma. Unlike the surrounding cell wall matrix it is resistant to “Onozuka” cellulase and remains intact during gibberellic acid-stimulated hydrolase release. After treatment of the tissue with gibberellic acid followed by digestion with “Onozuka” cellulase this resistant wall component can be isolated free of protoplast. Study of its surface features revealed the presence of numerous tubular extensions, 120 nm wide, connecting adjacent resistant walls. These tubes resembled light microscope images of plasmodesmata in size and appearance. E.M. sections of resistant walls showed the presence of unit membrane lining the inner surface of the wall tubes. It was concluded that the resistant wall constitutes a modified wall layer that is secreted uniformly across all plasmalemma surfaces, including those in the wall (plasmodesmata). The presence of wall tubes surrounding plasmodesmata enhances the apparent size of the plasmodesmata in the light microscope. This may account for previous inconsistencies in the literature between light and electron microscope determinations of plasmodesmata diameters.     

Ultrastructural pathology of leaf cells of ryegrass (Lolium multiflorum) infected with ryegrass mosaic virus.

Ryegrass mosaic virus particles and virus induced lamellar inclusions were found in mesophyll and epidermal cells of virus infected ryegrass leaves. The lamellar inclusions were occasionally found in phloem cells also. Virus particles occurred in cytoplasm, inside plasmodesmata and often in membrane bound sacs embedded in a matrix between plasmalemma and cell wall at or near plasmodesmata. Electron dense plugs protruding from plasmodesmata, finger-like cell wall outgrowths and cell wall deposits usually at plasmodesmata were also observed. Cytopathological changes in organelles in infected cells included dense deposits in the cisternae of endosplasmic reticulum and Golgi apparatus, mitochondria with electron-dense or opaque matrix, proliferating cristae and deteriorating unit membrane; and disintegrating chloroplasts.     

Modified plasmodesmata inSorghum (Sorghum bicolor L Moench) leaf tissues infected by maize dwarf mosaic virus

The early acute response (EAR), a type of hypersensitive response, is defined by small chlorotic spots at the base of the youngest leaf of sorghum (Sorghum bicolor L. Moench) cultivar HOK, and usually appears within five days after inoculation with maize dwarf mosaic virus strain A (MDMV-A). These chlorotic spots become necrotic one to two days later and the leaf tissues are rapidly killed. In leaf tissues showing EAR, plasmodesmal fields contained many modified plasmodesmata of various sizes and structures within thickened cell walls. The membranous vesicles and tubules, derived from the extended terminal structures of modified plasmodesmata, were blocked by callose-like deposits in the area between the cell wall and plasmalemma. Also observed were two opposite-directed channels united via a central cavity at the middle lamella of the cell wall, one end of which was connected to the plasmalemma, but the other end sealed off to form a bulbous extension. The localized structure, an extraprotoplasmic sac containing aggregates of elongated virus-like particles associated with the modified plasmodesmata, was located between the plasmalemma and the cell wall. The sac was bound by membranes, and appeared to be sealed and completely excluded from the protoplasm. Extraprotoplasmic sacs appeared to be derived from the terminal extension of modified plasmodesmata, and these modification seem to be related to restriction of the viral spread.     

Intramembrane changes occurring during maturation of herpes simplex virus type 1: freeze-fracture study.

During the maturation of two strains of herpes simplex virus type 1 (VR3 and Patton), intramembrane changes were detected with the freeze-fracture technique in the viral envelope and the infected cell plasma membrane, and these changes were compared with data obtained from thin sections. Regardless of the strain, the inner leaflet of the viral envelope of extracellular virions was characterized by a density of intramembrane particles (IMP) three times larger than the host nuclear and plasma membrane. Addition of IMP, which probably represent virus-coded proteins, was detected in the viral envelope only after budding from the nuclear membrane, whereas it occurred during envelopment of capsids at cytoplasmic vacuoles. Fused membranes also showed one of their fracture faces covered with a high density of IMP similar to that of the mature virion envelope. The internal side of the membrane leaflet bearing these numerous particles was always characterized by the presence of an electron-dense material in thin sections. In addition, the plasma membrane of fibroblasts and Vero cells showed strain-specific changes: patches of closely packed IMP were observed with the VR3 strain, whereas ridges almost devoid of IMP characterized the plasmalemma of cells infected with the Patton strain. These intramembrane changes, however, were not observed as early as herpes membrane antigens. Thus, application of the freeze-fracture technique to herpes simplex virus type 1-infected cells revealed striking structural differences between viral and uninfected cell membranes. These differences are probably related to insertion and clustering of virus-coded proteins in the hydrophobic part of the membrane bilayer.     

Eyespot membranes in newly released zoospores of the green algaChlorosarcinopsis gelatinosa (Chlorosarcinales) and their fate during zoospore settlement

Summary Eyespot membranes in zoospores of the green algaChlorosarcinopsis gelatinosa were studied with the freeze-fracture technique. The PF of the plasmalemma overlying the eyespot lipid globules contains significantly greater numbers of intramembraneous particles (IMP; 8,200 IMP/m²) compared to other areas of the plasmalemma (2,100 IMP/m²). In the eyespot area the EF of the plasmalemma reveals no IMP, but regularly arranged depressions corresponding to the PF particles. Sizes of PF particles are not significantly different between the eyespot area and other areas of the plasmalemma. Zoospore settlement starts approximately two hours after release and involves in sequence, rounding up of the cells, retraction of the flagella and secretion of a cell wall. Eyespot membrane specializations on the PF of the plasmalemma disappear during flagellar retraction and before cell wall secretion.The functional significance of eyespot membrane specializations is discussed in accordance with the view that these membranes are engaged in photoreception and primary sensory transduction relating to green algal phototaxis.     

A comparison of receptive and non-receptive plasma membrane areas of photoreceptor cells in the leech,Hirudo medicinalis

Summary Microvillar (receptive) and external (non-receptive) portions of the plasmalemma of photoreceptor cells of Hirudo were compared electron microscopically in thin sections and freeze-fracture replicas. A morphometric approximation showed that the surface area of the microvillar membrane is about 19 times larger than that of the external membrane. The microvillar membrane most probably undergoes extensive membrane turnover. In both segments of the membrane the particles associated with the P- and the E-fracture faces are randomly distributed except at some specific sites. The particles adhere predominantly to the P-faces. The particle densities on the fracture faces of the microvillar membrane differ from those of the external membrane. The P-face particles of the external membrane appear to be larger than those of the microvillar membrane. It is suggested that the P-face particles of the microvillar membrane represent sites where the photopigment is incorporated into the membrane. The distinguishing structural features correspond to the functional differences postulated for both portions of the plasma membrane.     

Regular arrays of intramembranous particles in the plasmalemma of guard cell and mesophyll cell protoplasts of Vicia faba

Freeze fractures of the plasmalemma membranes of guard-cell and mesophyll protoplasts of Vicia faba demonstrate that the inner monolayer of the plasmalemma is compartmentalized into areas with distinct, highly organized structures. Between areas of intramembranous particles dispersed randomly on a relatively smooth fracture face, membrane domains showing an extremely regular planar, hexagonal array of particles are interspersed. The dimensions of these hexagonal lattices are about 0.5 m in diameter, the center-to-center spacing is about 22 nm, and the particle size is about 9 nm. The particle in the hexagonal arrays are accompanied by complementary pits in the opposite monolayer fracture of the plasmalemma membrane.The freeze-fracture preparation was performed by using an improved Leybold Bioetch device which provides a sufficiently high cooling rate and allows the omission of cryoprotectants, like glycerol.Presented by H. Schnabl on the Workshop on Plant Membrane Transport, Toronto, Canada, July 1979     

Morphological features of the surface of the sea urchin (Arbacia punctulata) egg: Oolemma-cortical granule association

Scanning microscopy and transmission electron microscopy of sectioned specimens and freeze-fracture replicas revealed the presence of slightly elevated regions, approximately one-fourth to one-half the diameter of microvilli, which were situated along the surface of unfertilized Arbacia eggs. These modifications of the surface of the egg were observed in areas occupied by cortical granules and were greatly reduced in number following the cortical granule reaction. Few such modifications were present in immature and urethane-treated ova, in which cortical granules were located in regions of the egg other than the cortex. Freeze-fracture replicas of unfertilized eggs revealed a significantly higher density of intramembranous particles within the plasmalemma when compared to replicas of the membrane surrounding cortical granules. Areas characteristic of the cortical granule membrane, i.e., sparsely laden with particles, were not observed within the plasmalemma of the fertilized egg. Hence, following its fusion with the egg plasma membrane there is a dramatic reorganization in particle distribution of the membrane derived from cortical granules.     

Expression of the Kidney Bean Phenylalanine-Ammonia Lyase Gene in the Hairy Roots of Astragalus sinicus

Using Agrobacterium rhizogenes, Astragalus sinicus plants were transformed with the kidney bean pal5 gene coding for phenylalanine-ammonia lyase (PAL). The hairy root culture thus obtained manifested enhanced PAL activity and lignin content in the cell walls; in addition, the transformed cells differed from the wild-type ones in several electrophysiological indices. In particular, the diffusion component of the total membrane potential of plasmalemma increased in the pal-transformed roots. The authors presume that the volume density of the protein-related negative charge of the cytoplasm increases in the transformed root cells along with changes in the cytoplasmic pH and pCa²⁺, the extent of coupling of these two indices, and the hydraulic conductivity of plasmodesmata.     

LEAF OF SPINACIA OLERACEA (SPINACH): ULTRASTRUCTURE,AND PLASMODESMATAL DISTRIBUTION AND FREQUENCY,IN RELATION TO SIEVE-TUBE LOADING

Minor veins and contiguous tissues of the Spinacia oleracea leaf were analyzed by electron microscopy to determine the characteristics of the component cells and the structure, distribution, and frequency of plasmodesmata between the various cell types of the leaf. Mesophyll and bundle-sheath cells contain components typical of photosynthetic cells although the latter cell type contains smaller chloroplasts and fewer mitochondria and microbodies than the mesophyll cells. In addition, the mesophyll cells contain numerous invaginations of the plasmalemma bordering the chloroplasts and evaginations of the outer membrane of the opposing chloroplast envelope. In places, these membranes appear continuous with each other. The minor veins consist of tracheary elements, xylem parenchyma cells, sieve-tube members, companion and phloem parenchyma cells, and other cells simply designated vascular parenchyma cells. The companion and phloem parenchyma cells are typically larger than the sieve-tube members with the companion cells containing a much denser cytoplasm that the phloem parenchyma. Cytoplasmic connections occur along all possible routes from the mesophyll to the sieve-tube members and consist of either simple or branched plasmodesmata between parenchymatic elements or pore-plasmodesmata between the sieve-tube members and parenchyma cells. The highest frequency of plasmodesmata occurs between the sieve-tube members and companion cells, although the value is essentially the same as between the various parenchymatic elements of the phloem. Compared to several previously studied species, the frequency of plasmodesmata between cell types of the spinach leaf is low. These results are discussed in relation to apoplastic vs. symplastic solute transport and sieve-tube loading in this species.     

The neck constriction in plasmodesmata

Simple plasmodesmata between mesophyll and bundle sheath cells in actively expanding leaves of Salsola kali L. and roots of Epilobium hirsutum L. are shown to possess specialized structures, called sphincters, around their neck regions. The sphineters are made visible by the combined effects of tannic acid and heavy metal staining; they are localized just outside that area of the plasmalemma, which forms the collar around the entrance to each plasmodesmos. This localization corresponds to a very active area of the plasmodesmos/olasmalemma complex (i.e. enzyme activity and/or presence of strongly reducing substances).Evidence is presented that these ring structures are structural equivalents to hypothetical sphincters performing some valve function; i.e. participating in the control of rates and directions of symplastic transport of solutes through plasmodesmata. The middle layer of the plasmalemma in the neck region is composed of closely-packed, globular subunits appearing in negative contrast. Apparently, these subunits correspond to particle clusters observed at the plasmodesmatal entrance in freeze-fracture preparations. They appear similar to particle clusters in animal tight junctions, and their possible function in providing electrical coupling via low resistance junctions between plant cells is discussed.     

Freeze-fracture evidence of gel-phase lipid in membranes of senescing cowpea cotyledons

The structural details of membrane organization in germinating and senescing cotyledons of cowpea (Vigna unguiculata (L.) Walp.) were studied by thin section and freeze-fracture electron microscopy. Germination- and senescence-related changes in the ultrastructure of parenchymal cells of cowpea cotyledons, as detected in thin sections, closely resemble those described for other leguminous seeds. Additionally, electron-dense deposits associated with the membranes, particularly the plasmalemma and endoplasmic reticulum, were seen to increase with advancing senescence. Freeze-fracture electron microscopy demonstrated that the membranes of cotyledons of 2-d-old seedings appear to be normal, with evenly dispersed intramembranous particles. However by 4 d, small areas or domains of the plasmalemma were free of intramembranous particles. These particle-free areas increased in both size and number as senescence progressed. We interpret these particle-free areas to be structural evidence for lateral phase separations of the membrane lipids into microdomains of gel-phase lipid from which intrinsic membrane proteins are excluded. Our results support wide-angle X-ray diffraction studies which have demonstrated the presence of gel-phase lipids in senescing bean cotyledons.Abbreviations EF exoplasmic fracture - ER endoplasmic reticulum - ESR electron-spin resonance - IMP(s) intramembranous particle(s) - PF protoplasmic fracture     

Filament Disruption in Funaria Protonemata: Occlusion of Plasmodesmata

Plasmodesmata are occluded when Funaria chloronemata are fragmented by the development of tmema cells (TCs). The TC deposits a new wall layer along the cross wall toward the neighbouring non-sister cell (NC). This wall layer cuts off the plasmodesmata and its connection with the cross wall is soon lost. The plasmodesmata become isolated when the NC forms a new wall layer along the former cross wall. At the end of TC development, before its disintegration, the sister cell (SC) also deposits a new wall layer along the cross wall toward the TC, cutting off the plasmodesmata. For some time the plasmalemma of the plasmodesmata remains connected to the NC or the TC, whereas the desmotubule soon disappears. Relicts of the plasmalemma remain even after the isolation of the plasmodesmata and the disintegration of the TC. During the decay of the plasmodesmata, a cylinder of electron-dense material is frequently formed along the border of the plasmodesmatal channel. This may extend over the surface of the cell wall. Eventually, the plasmodesmatal channel is filled with wall material. Callose is only observed around functional plasmodesmata and does not seem to play a role in their occlusion.     

The Ultrastructure and Computer-enhanced Digital Image Analysis of Plasmodesmata at the Kranz Mesophyll-Bundle Sheath Interface of Themeda triandra var. imberbis (Retz) A. Camus in Conventionally-fixed Blades

The ultrastructure of the plasmodesmata at the Kranz mesophyll-bundlesheath (KMS-BS) interface in Themeda triandra, and the substructureswithin the plasmodesmata were investigated, using conventionallyfixed leaf-blade material, enhanced by the addition of 0·1%tannic acid to the primary fixative. Examination of high-resolution electronmicrographs, and computer-enhanceddigital images suggests that these plasmodesmata are complexstructures, comprised of helically-arranged particulate material.The electron-dense particles are between 2·5 and 3·0nm in diameter. These particles are specifically associatedwith the inner face of the inner plasmalemma membrane leaflet,and the outer region of the desmotubule wall. The electron-denseparticles are presumably proteinaceous and embedded in a lipidmatrix. In the constricted median portion of the KMS-BS plasmodesmata,the space between the desmotubule and the inner plasmalemmamembrane leaflet and areas surrounding the proteinaceous particlesthereof (the cytoplasmic sleeve) is about 3 nm in cross-section,and constitutes what we believe to be the space through whichintercellular transport takes place.Copyright 1993, 1999 AcademicPress Themeda triandra, Poaceae, cytoplasmic sleeve, image analysis, plasmodesmatal structure, desmotubule, Kranz mesophyll     

Isolation and properties of the plasmalemma in yeast

Summary A method is described for the isolation of fragments of the plasmalemma based on differential and density gradient centrifugation using cell free extracts from anaerobically grown Saccharomyces cerevisiae. Electron microscopically investigated frozen-etched specimens of isolated plasmalemma revealed the presence of globular particles attached to the outer surface of the membrane; these particles correspond to those observed in situ.In isolated plasmalemma a high specific activity of Mg⁺⁺-dependent ATPase, which is not sensitive to Oligomycin, is present. Yeast plasmalemma contains protein, lipids (including phospholipids) and an appreciable amount of polysaccharide. Hydrolysis of this polysacharide yields only mannose.The treatment of the isolated plasmalemma with detergents liberates the globular particles which can be isolated by density gradient centrifugation. Protein and polysaccharide occur in the respective fraction; therefore the globular particle represents a mannan-protein. It is concluded that the particles, which cover the plasma-membrane of plant cells, represent glycoproteins, that is, building stones to be incorporated into the fibrillar network of the cell walls.     

Asymmetric distribution of the plasma-membrane H+-ATPase in embryos of Vicia faba L. with special reference to transfer cells

The ultrastructural localization of the plasma-membrane H⁺ -ATPase by immunocytochemistry was studied in Vicia faba embryos which absorb nutrients from the maternal organism through the transfer cells of their external epidermis. The samples were embedded in LR White resin and the specificity of immunolabelling was checked by inhibition in the presence of purified H⁺-ATPase. The following results were obtained: (i) The H⁺-ATPase density varied according to the cell type, being higher in transfer cells than in other cell types, especially the non-modified cells of the internal epidermis. (ii) There was a marked polarity in transfer cells as proton pumps were more numerous in the area of plasmalemma infoldings where active nutrient uptake is assumed to take place, (iii) No clear immunolabelling occurred on the plasma membrane of plasmodesmata. These results demonstrate that in transfer cells the area of plasmalemma infoldings is highly specialized for active solute transport; they also support the idea of specific structural properties of the plasmalemma in plasmodesmata.This work was supported by the Centre National de la Recherche Scientifique (URA CNRS 574). We express our gratitude to Dr M.G. Palmgren (Royal Veterinary and Agricultural University, Copenhagen, Denmark) for his gift of purified H⁺-ATPase. We wish to thank J.C. Fromont for his skillful technical assistance with the immunological procedures. We are grateful to J.M. Perault and C. Besse of the Electron Microscopy Service (Service Universitaire de Microscopie Electronique Appliquée à la Biologie Poitiers, France) for their contribution to the microscopical techniques.     

毛竹茎秆纤维细胞发育过程中ATP酶的超微细胞化学定位研究

采用磷酸铅沉淀技术,对毛竹茎秆纤维细胞发育过程中的ATP酶进行了超微细胞化学定位研究.在初生壁形成时期,大量的ATP酶的活性产物沉积在质膜、质膜内陷、运输小泡、胞间连丝等膜体系以及细胞核和各种细胞器上;在次生壁形成的初期,ATP酶在多泡小体和裂解的液泡膜上出现,凝聚并边缘化的染色质上仍然具有ATP酶活性;随着次生壁的逐渐加厚,在前四年中持续存在具有ATP酶活性的质膜内陷结构,以后消失;而在六年生纤维细胞的质膜、运输小泡、纹孔、胞间连丝和凝聚化的染色质上仍然发现有明显的ATP酶分布,并发现在染色质上ATP酶活性会随着凝聚程度的加深而增强.结果表明,ATP酶在毛竹茎秆纤维细胞壁的整个形成过程中发挥重要作用,而纤维细胞的次生壁形成过程是一个由核基因控制的主动的PCD过程;并证实毛竹茎秆纤维细胞的发育有别于其它木本植物纤维细胞的发育过程,这种纤维细胞是一种典型的长寿细胞.     

Wall-to-membrane linkers in onion epidermis: some hypotheses

Wall-to-wall linkage may help maintain cell integrity and polarity, and focus mechanical stress from wall to mech-anotransductive ion channels within the plasm a lemma. When cells of onion bulb scale epidermis shrink during plasmolysis with CaCl₂, the plasmalemma remains attached to the cell wall by Hechtian strands which we hypothesize might possibly be drawn out from linkages fulfilling the above functions. We show that at least many of the attachment loci are independent of the plasmodesmata. A priori, wall glycoproteins seem good candidates for the wall-to-membrane linkers; therefore, we investigated the distribution in wall and plasmalemma of antigen recognized by antibody to hydroxyproline-rich glycoprotein (HRGP). Using fluorescent secondary antibodies, we showed that polyclonal antibodies prepared against wall HRGP from soybean bind to the onion walls (following mild depectination), but also bind to the plasmalemma after the wall is enzymatically digested. The distribution of the antibodies is punctate. On the plasmalemma, the points tend to be scattered more or less uniformly, but can cluster at termini of large streaming strands (which rarely form in wall-constrained cells.) These streaming strands can be seen to exert tension on the membrane. We hypothesize that (1) the antigen on the surface of the protoplast may correspond to the antigen in the walls, (2) such antigen may be responsible for adhesion of membrane to wall at the linkage sites visualized by CaCl₂ plasmolysis, and (3) the linkage sites may be transmembrane proteins to which cytoskeleton can attach at the inner surface.     

Freeze-Fracture Study of Malaria Sporozoites: Antibody-Induced Changes of the Pellicular Membrane*

Plasmodium cynomolgi, Plasmodium knowlesi, and Plasmodium berghei sporozoites, before and after incubation with immune serum, were studied after freeze-fracture by electron microscopy. There were evenly distributed numerous intramembranous particles (IMP) on the P face of the outer membrane. The E face of the plasma membrane had fewer IMP than its P face. The E face of the intermediate membrane had few IMP and also linear arrays of slightly raised ridges running the length of the parasite. The P face of the intermediate membrane had many IMP aligned along the long axis of the sporozoite. On the P face of the inner membrane. IMP were arranged in very distinct rows conforming to the long axis of the parasite; the E face of this membrane had a few randomly distributed IMP. A prominent change in the sporozoite incubated in immune serum was the appearance of a layer of aggregated particles around the parasite. The P face of the plasma membrane had several clear areas devoid of IMP and IMP aggregates. No changes were seen in the other fractured faces of the pellicle. These observations suggest that immune serum acts only on the P face of the plasma membrane.     

A freeze-etch study of the ultrastructure of red algal pit plugs

Summary Freeze-etch studies indicate that the fully-formed pit plug ofPalmaria palmata consists of a homogeneous plug core, a pair of plug caps, and associated membranes. A single cap membrane separates the two layers of each plug cap and is the only membrane found between the cytoplasm and the plug core. Due to the apparent junction of the plasmalemma and cap membranes, the plug core, along with the adjacent plug cap layer on either face of the core, is entirely membrane-bounded and essentially extracellular. Fracture faces of the cap membrane bear large populations of particles which are inconspicuous due to their low profile. The plasmalemma lining the plug core from cell to cell has numerous, very obvious particles on its fracture faces, more even than does the cytoplasmic plasmalemma.The relationship of the rhodophycean pit plug to the fungal septal pore plug is discussed.This report represents a portion of a thesis submitted in partial fulfillment of the requirements of a Master of Science degree, Cornell University, Ithaca, N.Y.