A correlated histochemical and ultrastructural study of the epidermis and hypodermis of onion roots

Summary Cell walls of mature epidermal and hypodermal cells are autofluorescent when viewed under ultraviolet or blue light. This autofluorescence develops in a centripetal direction, beginning in the outer tangential wall of the epidermis and ending in the inner tangential wall of the hypodermis. The intercellular regions between the epidermis and hypodermis and between the hypodermis and the cortex are dense and also become autofluorescent. Although the walls of the hypodermis provide a barrier to the movement of a high molecular weight fluorescent dye, the walls of the epidermis are permeable. Histochemical studies indicate that lipids and polyphenolics are components of the epidermal and hypodermal cell walls. Both layers are resistant to the wall-degrading enzyme Driselase and to concentrated sulphuric acid, whereas the cortex is digested with both treatments. Observations with the transmission electron microscope show that a complex suberin lamella encases each hypodermal cell but is absent from the epidermis. However, the outer tangential wall and radial walls of the epidermal cells are complex in that layers of different densities are present. Some of these layers, as well as the intercellular regions and the radial walls of the hypodermal cells, bind ferric ions when tissue is fixed in ferric chloride-glutaraldehyde indicating the presence of poly-phenolics in these regions. An extracellular layer covering the outer tangential wall of the epidermis stained positively with a number of histochemical tests for polyphenolics.     

THE STRUCTURE OF THE PRIMARY EPIDERMAL CELL WALL OF AVENA COLEOPTILES

The primary walls of epidermal cells in Avena coleoptiles ranging in length from 2 to 40 mm. have been studied in the electron and polarizing microscopes and by the low-angle scattering of x-rays. The outer walls of these cells are composed of multiple layers of cellulose microfibrils oriented longitudinally; initially the number of layers is between 10 and 15 but this increases to about 25 in older tissue. Where epidermal cells touch, these multiple layers fuse gradually into a primary wall of the normal type between cells. In these radial walls, the microfibrils are oriented transversely. Possible mechanisms for the growth of the multilayered outer wall during cell elongation are discussed.     

Seed coat development, anatomy and scanning electron microscopy of Harpagophytum procumbens (Devil's Claw), Pedaliaceae

Seed coat development of Harpagophytum procumbens (Devil's Claw) and the possible role of the mature seed coat in seed dormancy were studied by light microscopy (LM), transmission electron microscopy (TEM) and environmental scanning electron microscopy (ESEM). Very young ovules of H. procumbens have a single thick integument consisting of densely packed thin-walled parenchyma cells that are uniform in shape and size. During later developmental stages the parenchyma cells differentiate into 4 different zones. Zone 1 is the multi-layered inner epidermis of the single integument that eventually develops into a tough impenetrable covering that tightly encloses the embryo. The inner epidermis is delineated on the inside by a few layers of collapsed remnant endosperm cell wall layers and on the outside by remnant cell wall layers of zone 2, also called the middle layer. Together with the inner epidermis these remnant cell wall layers from collapsed cells may contribute towards seed coat impermeability. Zone 2 underneath the inner epidermis consists of large thin-walled parenchyma cells. Zone 3 is the sub-epidermal layers underneath the outer epidermis referred to as a hypodermis and zone 4 is the single outer seed coat epidermal layer. Both zones 3 and 4 develop unusual secondary wall thickenings. The primary cell walls of the outer epidermis and hypodermis disintegrated during the final stages of seed maturation, leaving only a scaffold of these secondary cell wall thickenings. In the mature seed coat the outer fibrillar seed coat consists of the outer epidermis and hypodermis and separates easily to reveal the dense, smooth inner epidermis of the seed coat. Outer epidermal and hypodermal wall thickenings develop over primary pit fields and arise from the deposition of secondary cell wall material in the form of alternative electron dense and electron lucent layers. ESEM studies showed that the outer epidermal and hypodermal seed coat layers are exceptionally hygroscopic. At 100% relative humidity within the ESEM chamber, drops of water readily condense on the seed surface and react in various ways with the seed coat components, resulting in the swelling and expansion of the wall thickenings. The flexible fibrous outer seed coat epidermis and hypodermis may enhance soil seed contact and retention of water, while the inner seed coat epidermis maintains structural and perhaps chemical seed dormancy due to the possible presence of inhibitors.     

Morphological alterations of cell wall concomitant with protein release in a protein-producing bacterium, Bacillus brevis 47.

Bacillus brevis 47 secreted vast amounts of protein into the medium and had a characteristic three-layered cell wall. The three layers are designated, from the outermost to the innermost layer, as the outer wall (4.2 nm), the middle wall(8.5 nm), and the inner wall (2.1-3.7 nm). The inner wall might be a peptidoglycan layer. The fine cell wall structure was morphologically altered to various extents, depending on the growth period. At the early stationary phase of growth, cells began to shed the outer two layers of a limited area of the surface. This shedding was complete after further cell growth. The morphological alterations in the cell wall occurred concomitantly with a prominent increase in protein excretion. When protein secretion was severely inhibited by growing cells with Mg2+, morphological alterations in the cell wall were not observed, even at the late stationary phase of growth. This was also the case with a nonprotein-producing mutant, strain 47-5-25. When cells were incubated in buffers, the outer two layers of the cell wall were specifically removed, leaving cells surrounded only by the inner wall layer. The layers removed by incubation were recovered by high-speed centrifugation. This fraction consisted of two layers resembling the outer and middle wall layers. Protein secreted by B. brevis 47-5 consisted mainly of two proteins with approximate molecular weights of 150,000 and 130,000. Proteins released by incubating cells in buffers and proteins in the outer- and middle-wall-enriched fraction were also composed mainly of two proteins with the same molecular weights as those secreted into the medium. Therefore, we conclude that B. brevis 47 secretes proteins derived from the outer two layers of cell wall and these components are synthesized even after the shedding of the outer two layers.     

Demonstration by freeze-etching of a single cleavage plane in the cell wall of a gram-negative bacterium

In the examination of protoplasts of a gram-negative bacterium classified as a Pseudomonas sp. by freeze-etching, we found a smooth external surface which is not seen if the preparations are not "etched." This external structure is seen as a sleeve surrounding and connecting the cells in unetched preparations, and we present evidence that it is a eutectic formed during the freezing of the specimen. In the system used in this study, the four layers of the cell wall of a gram-negative bacterium can be removed from the cell. The single cell wall cleavage plane is not affected by the removal of the loosely bound outer layer or of the peptidoglycan layer, but it is lost when the outer double track layer and the underlying soluble layer are simultaneously removed. Thus, we conclude that it is one of these two layers which is responsible for the cleavage plane which exposes variable areas of a smooth surface in the cell wall. This cell wall cleavage plane is more likely to deflect the actual cleavage of the frozen cell when cells are relatively old or when they are suspended in sucrose.     

STRUCTURE AND DEVELOPMENT OF WALLS IN FUNARIA STOMATA

The development and structure of the guard cell walls of Funaria hygrometrica Hedw. (Musci) were studied with the light and electron microscopes. The stoma consists of only one, binucleate guard cell as the pore wall does not extend to the ends of the cell. The guard cell wall is thinnest in the dorsal wall near the outer wall but during movement is most likely to flex at thin areas of the outer and ventral walls. The mature wall contains a mottled layer sandwiched between two, more fibrillar layers. The internal wall layer has sublayers with fibrils in axial and radial orientations with respect to the pore. During substomatal cavity formation, the middle lamella is stretched into an electron dense network and into strands and sheets. After stomatal pore formation, the subsidiary cell walls close to the guard cell become strikingly thickened. The functional implications of these results are discussed.     

ULTRASTRUCTURE AND DEVELOPMENT OF THE MICROSPORANGIUM OF PSEUDOTSUGA MENZIESII (MIRB.) FRANCO. I. DORMANT STAGES

The dormant (mid-November to mid-February) microsporangia of Pseudotsuga menziesii (Douglas-fir) contain pollen mother cells (PMC's) in diffuse diplotene, surrounded by 1–2 layers of tapetal cells and 3–4 layers of microsporangial wall cells. At the beginning of dormancy, PMC's are large and their walls are lysed. The cell walls contain a thick layer of loosely-arranged fibrils which are produced in large vesicles in the PMC cytoplasm and are secreted across the plasma membrane. PMC's contain several layers of rough ER. The inner tangential and the radial walls of the tapetal cells are lysed. During dormancy the PMC's form many new autophagic vacuoles, the chromatin consists of a network of fine threads comprised of medium-sized granules of uniform size and the nucleoli split. The outer tapetal wall is thick and becomes encrusted by an irregular lipid layer. The tapetal cytoplasm is similar to the PMC cytoplasm but is devoid of amyloplasts. The tapetal cytoplasm shows secretory activity at the beginning of dormancy and again near the end of dormancy. The later secretory activity results in the deposition of a spongy material, especially along the radial and inner walls of the tapetal cells. Tapetal cells contain 1–2 large nuclei which show prominent and irregular clumps of chromatin. Subcellular developmental changes occur in the dormant microsporangia of Pseudotsuga in much the same manner as has been reported for Pinus.     

Ultrastructural and Chemical Alteration of the Cell Envelope of Pseudomonas aeruginosa, Associated with Resistance to Ethylenediaminetetraacetate Resulting from Growth in a Mg2+-Deficient Medium

Cells of Pseudomonas aeruginosa became resistant to the lytic effect of ethylenediametetraacetate (EDTA) when grown in a Mg(2+)-deficient medium. To correlate ultrastructural changes in the cell wall associated with the shift to EDTA-resistance, a freeze-etch study was performed. Upon fracturing, the outer cell wall membrane split down the hydrophobic center to reveal the outer (concave) and inner (convex) layers. The concave cell wall layer of EDTA-sensitive cells grown in Mg(2+)-sufficient medium contained spherical units resting on an underlying smooth support layer. Upon EDTA treatment, approximately one-half of these spherical units were extracted. Cells grown in Mg(2+)-deficient medium were resistant to EDTA. The concave cell wall layer of EDTA-resistant cells had increased numbers of highly compacted spherical units, giving this layer a disorganized appearance. The highly compacted appearance of this layer was unaltered by EDTA treatment. Thus, growth in Mg(2+)-deficient medium resulted in cells which were resistant to EDTA and which possessed an ultrastructurally altered outer layer of the outer cell wall membrane. Cell envelopes from EDTA-resistant cells were found to possess 18% less phosphorus, 16.4% more total carbohydrate, and 13.3% more 2-keto-3-deoxyoctonate than cell envelopes from EDTA-sensitive cells. There were also qualitative, but not quantitative, differences in the protein content of cell envelopes from EDTA-resistant and EDTA-sensitive cells.     

Location of the Fracture Faces Within the Cell Envelope of Acinetobacter Species Strain MJT/F5/5

The cell wall of the gram-negative bacterium Acinetobacter species strain MJT/F5/5 shows in thin section an external “additional” layer, an outer membrane, an intermediate layer, and a dense layer. Negatively stained preparations showed that the additional layer is composed of hexagonally arranged subunits. In glycerol-treated preparations, freeze-etching revealed that the cell walls consist of four layers, with the main plane of fracture between layers cw 2 and cw 3. The surface of [Formula: see text] 2 consisted of densely packed particles, whereas [Formula: see text] 3 appeared to be fibrillar. In cell envelopes treated with lysozyme by various methods, the removal of the dense layer has detached the outer membrane and additional layer from the underlying layers, as shown in thin sections. When freeze-etched in the absence of glycerol, these detached outer membranes with additional layers fractured to reveal both the faces [Formula: see text] 2 and [Formula: see text] 3 with their characteristic surface structures, and, in addition, both the external and internal etched surfaces were revealed. This experiment provided conclusive evidence that the main fracture plane in the cell wall lies within the interior of the outer membrane. This and other evidence showed that the corresponding layers in thin sections and freeze-etched preparations are: the additional layer, cw 1; the outer membrane, cw (2 + 3); and the intermediate and dense layers together from cw 4. Because of similarities in structure between this Acinetobacter and other gram-negative bacteria, it seemed probable that the interior of the outer membrane is the plane most liable to fracture in the cell walls of most gram-negative bacteria.     

Ontogeny and structure of the drupe of Ozoroa paniculosa (Anacardiaceae)

An exocarp sensu stricto develops from the outer epidermis of the ovary wall. At maturity it comprises extensively radially elongated palisade-like parenchyma cellS. Besides having an outer cuticle, the outer tangential and outer parts of the radial cell walls of these cells are strongly cutinized. Large, permanently open stomata and saucer-shaped depressions also characterize the exocarp. The mature mesocarp sensu stricto consists of secondarily thickened parenchyma and brachysclereidS. An abundance of tanniniferous deposits and crystals, as well as secretory ducts associated with the vascular bundles also form part of the mature mesocarp. Derivatives of the inner epidermis of the ovary wall differentiate into the stratified endocarp sensu stricto. At maturity this comprises consecutive layers of macrosclereids, osteosclereids (typified by a capitate part and cell wall flutes), brachysclereids, and crystalliferous sclereidS. Pericarp structure is related to its taxonomic significance and the possible role of micromorphological characters in the survival strategy of Ozoroa paniculosa. It is shown that ontogenetic studies contribute to the precise interpretation of previously described cell layers, ensuring that homologous tissues are compared in different taxa.     

Electron Microscopic Observations on the Structure of the Envelopes of Mature Elementary Bodies and Developmental Reticulate Forms of Chlamydia psittaci

Purified suspensions of Chlamydia psittaci were prepared from L cells. Thin sections of intact elementary bodies and intact developmental reticulate bodies and of their purified envelopes were observed by electron microscopy. In both intact organisms and partially purified envelopes, two membranous structures, each appearing in electron micrographs as two darkly stained layers, were observed. In the elementary body sections, the outer membrane was round, apparently rigid, and was not soluble in 0.5% sodium dodecyl sulfate. The inner layer was irregular in shape and was completely removed by detergent treatment. We interpret these results to indicate that the outer rigid layer of the envelope is the cell wall and the inner layer is the cytoplasmic membrane. When the fragile reticulate body envelopes were similarly studied, the outer cell wall was clearly visible, and some evidence of an inner membrane was seen. After treatment with nucleases and detergent, all evidence of inner or cytoplasmic membrane was removed, but the outer cell wall remained. Thus, it appears that the cell wall of this organism is continuous throughout the growth cycle and that the fragility and lack of rigidity of the reticulate body cell is due to changes in chemical composition or structure of the cell wall.     

The formation of wall-like envelopes by isolated tomato-fruit protoplasts

Summary Formation of a new cell wall around tomato protoplasts was confirmed by optical microscopy, electron microscopy and X-ray diffraction. This wall is composed of three layers; (a) an outer ring, which seems to be composed of diffuse, amorphous material, (b) an intermediate space, crossed by radial fibers, (c) a thicker, inner band composed of dense, highly consolidated material which may have sub-layers within it. Occasionally, cells are observed with only the dense consolidated layer about them. The origin of this wall and its component layers is not yet understood.National Research Council Post-doctoral Fellow, 1967–1969.     

Organization of mature external glands on the trap and other organs of the bladderwortUtricularia monanthos

Summary The mature dome-shaped glands which cover the outer surfaces of the trap, leaves, anchor and runner stolons inU. monanthos are described using conventional and some high voltage transmission electron microscopy. The glands occur as scattered ordinary external glands and as a compact clump of vestibule glands at the entrance to the doorway. Each gland rests on a basal epidermal cell and consists of a single pedestal and terminal cell. Vestibule and leaf glands differ slightly from the other glands mainly in the structure of the outer wall of the terminal cell. Nuclear crystals are prominent and the cytoplasm of the pedestal and terminal cells contains tubular structures usually aggregated near the nucleus. The pedestal cell is a transfer cell with short wall protuberances on the outer wall, conspicuous mitochondria and a heavily impregnated lateral wall.The terminal cell often has an outer wall that is greatly thickened and a protoplast that may degenerate early. In the most developed cells the protoplast remains active for a long period and the outer wall is differentiated into several layers. The outermost layer is cuticularized consisting of an open meshwork of deposits. In leaf glands a local polysaccharide mass is usually developed within the cuticularized region. The inner non-impregnated region of the outer wall may show four layers. In vestibule glands fewer layers are present and the wall shows prominent lamellations. Some ordinary external glands differentiate a sponge-like substructure within the inner wall.The ultrastructure and function of the glands are discussed. We support the concept that mature external glands are responsible for secreting water, with those on traps being particularly active during the resetting of the organ. Our work provides a structural basis for recent suggestions by other workers that the mechanism of secretion probably involves establishing a standing osmotic gradient within the gland.     

Relationship of a Wall-Associated Enzyme with Specific Layers of the Cell Wall of a Gram-Negative Bacterium

In untreated cells of the marine pseudomonad studied here, alkaline phosphatase was found to be located in the periplasmic space, at the cell surface, and in the medium into which it had been shed during growth. Washing in 0.5 M NaCl, which removed the loosely bound outer layer, caused a shift of periplasmic enzyme to the outer aspect of the double-track layer and released some of the cell surface-associated enzyme. When the double-track layer of the cell wall was partially deranged, large amounts of this cell wall-associated enzyme were released, and, when the double-track was removed from the cells to produce mureinoplasts, alkaline phosphatase was released into the menstruum. There was no significant association of the enzyme with the peptidoglycan layer of the cell wall, which is the outermost structure of the mureinoplast, and no association of the enzyme with the cytoplasmic membrane of these modified cells. This study has shown that alkaline phosphatase is specifically associated with the outer layers of the cell walls of cells of this organism and is retained within the cell wall by virtue of this association.     

Superficial Macromolecular Arrays on the Cell Wall of Spirillum putridiconchylium

Electron microscopy of the cell envelope of Spirillum putridiconchylium, using negatively stained, thin-sectioned, and replicated freeze-etched preparations, showed two superficial wall layers forming a complex macromolecular pattern on the external surface. The outer structured layer was a linear array of particles overlying an inner tetragonal array of larger subunits. They were associated in a very regular fashion, and the complex was bonded to the outer, pitted surface of the lipopolysaccharide tripartite layer of the cell wall. The relationship of the components of the two structured layers was resolved with the aid of optical diffraction, combined with image filtering and reconstruction and linear and rotary integration techniques. The outer structural layer consisted of spherical 1.5-nm units set in double lines determined by the size and arrangement of 6- by 3-nm inner structural layer subunits, which bore one outer structural layer unit on each outer corner. The total effect of this arrangement was a double-ridged linear structure that was evident in surface replicas and negatively stained fragments of the whole wall. The packing of these units was not square but skewed by 2 degrees off the perpendicular so that the "unit array" described by optical diffraction and linear integration appeared to be a deformed tetragon. The verity of the model was checked by using a photographically reduced image to produce an optical diffraction pattern for comparison with that of the actual layers. The correspondence was nearly perfect.     

Reciprocal influence of ethylene and gibberellins on response-gene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A cortical band of fiber cells originate de novo in tendrils of redvine [Brunnichia ovata (Walt.) Shiners] when these convert from straight, supple young filaments to stiffened coiled structures in response to touch stimulation. We have analyzed the cell walls of these fibers by in situ localization techniques to determine their composition and possible role(s) in the coiling process. The fiber cell wall consists of a primary cell wall and two lignified secondary wall layers (S₁ and S₂) and a less lignified gelatinous (G) layer proximal to the plasmalemma. Compositionally, the fibers are sharply distinct from surrounding parenchyma as determined by antibody and affinity probes. The fiber cell walls are highly enriched in cellulose, callose and xylan but contain no homogalacturonan, either esterified or de-esterified. Rhamnogalacturonan-I (RG-I) epitopes are not detected in the S layers, although they are in both the gelatinous layer and primary wall, indicating a further restriction of RG-I in the fiber cells. Lignin is concentrated in the secondary wall layers of the fiber and the compound middle lamellae/primary cell wall but is absent from the gelatinous layer. Our observations indicate that these fibers play a central role in tendril function, not only in stabilizing its final shape after coiling but also generating the tensile strength responsible for the coiling. This theory is further substantiated by the absence of gelatinous layers in the fibers of the rare tendrils that fail to coil. These data indicate that gelatinous-type fibers are responsible for the coiling of redvine tendrils and a number of other tendrils and vines.     

Egg release and germling development in Sargassum horneri (Fucales, Phaeophyceae)

The mature female conceptacle of Sargassum horneri (Turner) C. Agardh has an ostiole filled with a gelatinous plug. The oogonium in the conceptacle has cell walls that can be differentiated into a dense outer and a less dense inner microfibrillar layer. Just prior to egg release, stalk material is produced inside the outer layer and the inner layer disappears. At this stage the gelatinous plug is extruded and mucilage is released through the ostiole. The released eggs are retained on the receptacle by the stalk and are surrounded by a large amount of the mucilage. Three-celled germlings form a primary wall with a polylamellated structure of microfibril layers. In multicellular germlings that have differentiated into thallus and rhizoids, the peripheral thallus cells have an outer cell wall consisting of a microfibril layer under the primary wall, while the cell wall of the rhizoid tip has an amorphous structure. The germlings are released from the stalk and become attached to the substratum by an adhesive substance secreted from rhizoidal cells.     

Interaction of Mg-2+ with peptidoglycan and its relation to the prevention of lysis of a marine pseudomonad.

Intact cells of marine pseudomonad B-16 (ATCC 19855) which have been washed with a solution of NaCl require only 0.001 M MgSO4 and 100 to 300 times this concentration of NaCl or KCl to prevent lysis. Conversion of intact cells to mureinoplasts, a process involving removal of the outer double-track layer (outer membrane) and the periplasmic space layer of the cell wall, approximately doubled the requirement for the three salts to prevent lysis. The formation of protoplasts from mureinoplasts by removing the peptidoglycan layer again doubled the requirement for Na+ and K+ salts but increased the requirement for the Mg-2+ salt 200- to 300-fold. Cells of the marine pseudomonad suspended in solutions containing Mg-2+ salts failed to lyse on subsequent repeated suspension in distilled water, whereas cells presuspended in NaCl lysed immediately. Isolated envelope layers including the peptidoglycan layer, when dialyzed against solutiions containing Mg-2+ salts, retained Mg-2+ after subsequent suspension in distilled water. Envelope layers exposed to solutions of Na+ or K+ salts failed to retain these ions after exposure to distilled water. Na+ displaced Mg-2+ from the cell envelope layers. The results obtained indicate that the capacity of Mg-2+ salts at very low concentration to prevent lysis of intact cells and mureinoplasts of this organism is due primarily to the interaction of Mg-2+ with the peptidoglycan layer of the cell wall. Ion interaction with the layers lying outside of the peptidoglycan layer contributes only a small amount to the mechanical strength of the wall.     

Ultrastructural Explanation for Snapping Postfission Movements in Arthrobacter crystallopoietes

The ultrastructure of dividing rod-stage cells of Arthrobacter crystallopoietes was examined by electron microscopy. The cell walls consist of two layers. During cell division, the inner layer invaginates to form the septum. The outer layer does not participate in septum formation. After septum formation is completed, the two daughter cells remain attached by the outer layer of the cell wall. It appears that localized rupture of the outer layer during further wall growth is responsible for the phenomenon known as "snapping division" or "snapping postfission movement."