Structural and Biochemical Analysis of the Sheath of Phormidium uncinatum

The sheath of the filamentous, gliding cyanobacterium Phormidium uncinatum was studied by using light and electron microscopy. In thin sections and freeze fractures the sheath was found to be composed of helically arranged carbohydrate fibrils, 4 to 7 nm in diameter, which showed a substantial degree of crystallinity. As in all other examined motile cyanobacteria, the arrangement of the sheath fibrils correlates with the motion of the filaments during gliding motility; i.e., the fibrils formed a right-handed helix in clockwise-rotating species and a left-handed helix in counterclockwise-rotating species and were radially arranged in nonrotating cyanobacteria. Since sheaths could only be found in old immotile cultures, the arrangement seems to depend on the process of formation and attachment of sheath fibrils to the cell surface rather than on shear forces created by the locomotion of the filaments. As the sheath in P. uncinatum directly contacts the cell surface via the previously identified surface fibril forming glycoprotein oscillin (E. Hoiczyk and W. Baumeister, Mol. Microbiol. 26:699–708, 1997), it seems reasonable that similar surface glycoproteins act as platforms for the assembly and attachment of the sheaths in cyanobacteria. In P. uncinatum the sheath makes up approximately 21% of the total dry weight of old cultures and consists only of neutral sugars. Staining reactions and X-ray diffraction analysis suggested that the fibrillar component is a homoglucan that is very similar but not identical to cellulose which is cross-linked by the other detected monosaccharides. Both the chemical composition and the rigid highly ordered structure clearly distinguish the sheaths from the slime secreted by the filaments during gliding motility.     

Detection of a sheath on <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> polar flagellum

The presence of a polysaccharide sheath on the surface of the polar flagellum of Azospirillum brasilense was revelted by immunoelectron microscopy and immunodiffusion analysis with strain-specific antibodies to lipopolysaccharides (LPS). The antigenic identity of A. brasilense Sp245 sheath material and one of the two O-specific polysaccharides of its somatic LPS was demonstrated. The screening effect of the sheath in respect to flagellin was determined by agglutination tests and by the inhibition of azospirilla motility in liquid and semisolid agarized media caused by strain-specific antibodies to LPS; no pronounced effect of genus-specific antibodies to flagellin was observed.     

Characterization and Implications of the Cell Surface Reactivity of Calothrix sp. Strain KC97

The cell surface reactivity of the cyanobacterium Calothrix sp. strain KC97, an isolate from the Krisuvik hot spring, Iceland, was investigated in terms of its proton binding behavior and charge characteristics by using acid-base titrations, electrophoretic mobility analysis, and transmission electron microscopy. Analysis of titration data with the linear programming optimization method showed that intact filaments were dominated by surface proton binding sites inferred to be carboxyl groups (acid dissociation constants [pK_a] between 5.0 and 6.2) and amine groups (mean pK_a of 8.9). Sheath material isolated by using lysozyme and sodium dodecyl sulfate generated pK_a spectra similarly dominated by carboxyls (pK_a of 4.6 to 6.1) and amines (pK_a of 8.1 to 9.2). In both intact filaments and isolated sheath material, the lower ligand concentrations at mid-pK_a values were ascribed to phosphoryl groups. Whole filaments and isolated sheath material displayed total reactive-site densities of 80.3 × 10⁻⁵ and 12.3 × 10⁻⁵ mol/g (dry mass) of cyanobacteria, respectively, implying that much of the surface reactivity of this microorganism is located on the cell wall and not the sheath. This is corroborated by electrophoretic mobility measurements that showed that the sheath has a net neutral charge at mid-pHs. In contrast, unsheathed cells exhibited a stronger negative-charge characteristic. Additionally, transmission electron microscopy analysis of ultrathin sections stained with heavy metals further demonstrated that most of the reactive binding sites are located upon the cell wall. Thus, the cell surface reactivity of Calothrix sp. strain KC97 can be described as a dual layer composed of a highly reactive cell wall enclosed within a poorly reactive sheath.     

The role of the fibrillar component of the surface sheath in the morphogenesis of Dictyostelium discoideum

We have isolated a fibrillar component of the surface sheath of Dictyostelium discoideum by virtue of its insolubility in 9 M urea-2% sodium dodecyl sulfate (US). The US-insoluble material is primarily composed of cellulose, but also contains other carbohydrate components, protein, and lipid. Evidence is presented that the US-insoluble material is a component of the sheath. Sheath isolated from mutant strains lacking the developmentally regulated N-acetylglucosaminidase (NAG), α-mannosidase, or β-glucosidase activities is similar in composition to sheath isolated from wildtype strain. Strains lacking NAG are unable to migrate normally. This may result from the markedly lower crystallinity of the cellulose in the US-insoluble sheath isolated from these mutants. Strains lacking α-mannosidase or β-glucosidase migrate normally and the crystallinity of the sheath cellulose is not significantly below that of the wildtype. The correlation between the lower crystallinity of cellulose and the inability of strains lacking NAG to migrate suggests that crystallinity is physiologically important and that the degree of crystallinity is controlled by an enzymatic, mutable process. Strains U1 and UN1, which have <1% of the wildtype activity of uridine diphosphate glucose pyrophosphorylase activity, develop in a morphologically normal fashion to the slug stage. However, they cannot form stalk or spore cells, nor can they produce cellulose. These strains do not produce any detectable US-insoluble sheath and are fragile and unable to migrate. A continuous, nonfibrillar sheath surrounds the aggregates and is sufficient for normal morphogenesis up to the slug stage. The fibrillar component gives the aggregate the added rigidity required during migration.     

Comparative structural characterization of material extractable from aerial sheath of Streptomyces roseoflavus var. roseofungini

1. Tubular-like structures were regularly revealed in the surface sheath of the aerial mycelium of the parent strain of Streptomycetes roseoflavus var. roseofungini. In their shape and dimensions these structures were highly reminiscent of those massive accumulation of which was earlier reported to occur in cultures of dedifferentiated nocardioform “fructose” mutant of the same parent strain.
2. The tubular-like structures of the aerial mycelium sheath were shown to be markedly susceptible to brief acetone washing, undergoing almost complete desintegration.
3. On addition of water to crude acetone extract of the aerial mycelium precipitation and possible selfassembly of a spectrum of various structures occurred (folded scaly, bubble-like, spout-like). Some among structures so produced were reminiscent of those found in spectra of structures observed in reconstruction experiments with tubules from the mutant as well as of structures found in the aerial sheath of the parent strain and some other actinomycetes. Similarity in subunit structure of above materials was also noticeable.

The presence, in the sheath of aerial mycelium, of material with a tendency to selforganization is discussed in relation to the possible involvement of selfassembly processes in the formation of the surface sheath.     

Comparative development of the rattan ocrea,a structural innovation that facilitates ant–plant mutualism

The ocrea is an extension of the leaf sheath that occurs in a wide range of angiosperms, including some rattan palms (Arecaceae/Palmae). In some rattan species, the ocrea is an inflated sac-like structure that acts as a domatium. Typically, ants occupy this domatium, tending their young and husbanding aphids, and potentially providing a defence mechanism for the rattan. We present a comparative study of early leaf development in five palm species, including both ocreate and non-ocreate taxa. Early leaf development was examined using SEM in Calamus longipinna, which has a pronounced ocrea, and compared with two other Calamus species with an inconspicuous ocrea, and two non-ocreate palms, Chamaedorea pochutlensis and Rhapis humilis. All three Calamus species examined develop an extension from the top of the leaf sheath, in contrast with the non-ocreate palms examined for comparison. The ocrea is a vascularized outgrowth from the top of the leaf sheath, initiated shortly after differentiation of the lamina and sheath. Calamus longipinna differs from the other two Calamus species in that plications develop on the surface of the ocrea, and persist as folds on the ocrea surface. The highly unusual ocreate plications of C. longipinna form axial folds that permit the sac-like ocrea to become inflated, and give it the properties of a domatium, thus facilitating the ant–plant interaction in this species. The homologies of the ocrea in rattans require further review because the interpretation of leaf-derived appendages such as ligules and stipules remains controversial based on contradictory evidence from location and vasculature.     

Control of phototactic migration in Dictyostelium discoideum

Phototactic migration of pseudoplasmodia of the cellular slime mold, Dictyostelium discoideum, is directed by a response at the anterior tip. Horizontal light appears to be focused by refraction at the surface of the pseudoplasmodium such that it acts preferentially on the distal cells. We have been able to show that light stimulates the rate of pseudoplasmodial movement up to 80%. This increase is dependent on the intensity of the incident light. Thus it appears that light can control the direction of migration by increasing the rate of movement on the distal side. The anterior cells are then turned toward the light by cohesion to the more slowly moving proximal side. Migration rate in the dark may be limited by the rate of synthesis or deposition of the surface sheath surrounding the pseudoplasmodium. It is suggested that light increases the rate of migration by stimulating the formation of the surface sheath. Localized stimulation would then result in a turning response.     

Some Observations on the Fine Structure of the Giant Nerve Fibers of the Earthworm, Eisenia foetida

Sectioned dorsal giant fibers of the earthworm Eisenia foetida have been studied with the electron microscope. The giant axon is surrounded by a Schwannian sheath in which the lamellae are arranged spirally. They can be traced from the outer surface of the Schwann cell to the axon-Schwann membranes. Irregularities in the spiral arrangement are frequently observed. Desmosome-like attachment areas occur on the giant fiber nerve sheath. These structures appear to be arranged bilaterally in columns which are oriented slightly obliquely to the long axis of the giant fiber and aligned linearly from the axon to the periphery of the sheath. At these sites they bind together apposing portions of Schwann cell membrane comprising the sheath. Longitudinal or oblique sections of the nerve sheath attachment areas are reminiscent of the Schmidt-Lantermann clefts of vertebrate peripheral nerve. Septa of the giant fibers have been examined. They are symmetrical or non-polarized and consist of the two plasma membranes of adjacent nerve units. Characteristic vesicular and tubular structures are associated with both cytoplasmic surfaces of these septa.     

Monoclonal antibody characterisation of slime sheath: the extracellular matrix of Dictyostelium discoideum

Proteins can be extracted from the slime sheath of Dictyostelium discoideum slugs by denaturing agents. A subset of these proteins is also released by cellulase digestion of the sheath, implying that protein-protein and protein-cellulose interactions are involved in sheath protein retention. It seems probable that the cellulose-associated sheath proteins are also associated with the cellulose of mature stalk cells. Monoclonal antibodies directed against sheath demonstrate extensive sharing of antigenic determinants between sheath proteins and a limited degree of antigenic sharing between sheath and slug cell proteins. All of the proteins recognised by these monoclonal antibodies are developmentally regulated. These results are discussed in terms of the structure of the sheath and its possible role(s) in D. discoideum development.     

Separation of mesophyll protoplasts and bundle sheath cells from maize leaves for photosynthetic studies

Mesophyll protoplasts and bundle sheath strands of maize (Zea mays L.) leaves have been isolated by enzymatic digestion with cellulase. Mesophyll protoplasts, enzymatically released from maize leaf segments, were further purified by use of a polyethylene glycol-dextran liquid-liquid two phase system. Bundle sheath strands released from the leaf segments were isolated using filtration techniques. Light and electron microscopy show separation of the mesophyll cell protoplasts from bundle sheath strands. Two varieties of maize isolated mesophyll protoplasts had chlorophyll a/b ratios of 3.1 and 3.3, whereas isolated bundle sheath strands had chlorophyll a/b ratios of 6.2 and 6.6. Based on the chlorophyll a/b ratios in mesophyll protoplasts, bundle sheath cells, and whole leaf extracts, approximately 60% of the chlorophyll in the maize leaves would be in mesophyll cells and 40% in bundle sheath cells. The purity of the preparations was also evident from the exclusive localization of phosphopyruvate carboxylase (EC 4.1.1.31) and NADP-dependent malate dehydrogenase (EC 1.1.1) in mesophyll cells and ribulose 1,5-diphosphate carboxylase (EC 4.1.1.39), phosphoribulokinase (EC 2.7.1.19), and “malic enzyme” (EC 1.1.1.40) in bundle sheath cells. NADP-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) was found in both mesophyll and bundle sheath cells, while ribose 5-phosphate isomerase (EC 5.3.1.6) was primarily found in bundle sheath cells. In comparison to the enzyme activities in the whole leaf extract, there was about 90% recovery of the mesophyll enzymes and 65% recovery of the bundle sheath enzymes in the cellular preparations.     

Leaf and inflorescence peduncle anatomy: a contribution to the taxonomy of Rapateaceae

The anatomy of leaves and inflorescence peduncles was studied in species of Monotrema (4), Stegolepis (1) and Saxofridericia (1), aiming to contribute to the taxonomy of Rapateaceae. The form and structure of leaf blade midrib and the form of the inflorescence peduncle are diagnostic characteristics for the studied species. Monotrema is distinguished by: epidermal and vascular bundle outer sheath cells containing phenolic compounds in both organs; leaf blade with palisade and spongy chlorenchyma, arm-parenchyma, and air canals between the vascular bundles; leaf sheath with phenolic idioblasts in the mesophyll; inflorescence peduncle with tabular epidermal cells and air canals in the cortex and pith. Such characteristics support the recognition of Monotremoideae, which includes Monotrema. Stegolepis guianensis is distinguished by thick-walled epidermal cells and a plicate chlorenchyma in both organs; leaf blade with subepidermal fiber strands in abaxial surface and a heterogeneous mesophyll; inflorescence peduncle with rounded epidermal cells, a hypodermis with slightly thick-walled cells, and a pith with isodiametric cells and vascular bundles. Saxofridericia aculeata is distinguished by papillate epidermal cells in both organs; unifacial leaf blade with subepidermal fiber strands in both surfaces and a regular chlorenchyma; leaf sheath with a hypodermis in both surfaces and fiber bundles in the mesophyll; inflorescence peduncle with an undefined cortex and a hypodermis with thick-walled cells. S. guianensis shares few characteristics with S. aculeata, supporting their placement in different tribes.     

Mouthparts and stylet penetration of the lac insect Kerria lacca (Kerr) (Hemiptera:Tachardiidae)

Hitherto less known aspects on mouthpart morphology and penetration mechanism of the lac insect Kerria lacca have been explored. Unique details of the mouthparts, i.e. morphology of labium and stylets and salivary sheath have been brought out. The gross morphology of the mouthparts though resembled other plant sucking homopterans; a two-segmented labium with symmetrically distributed six pairs of contact-chemoreceptors on its surface was distinct; the mandibular stylets had serrations on its extreme apical region, while the maxillary stylets had their external surface smooth with parallel longitudinal grooves on their inner surface. Formation of flanges, salivary sheath and penetration pathway observed along with probing and penetration of the stylets intracellularly up to the phloem cells, as illustrated herein, are the addition to the existing knowledge on the structural details of the mouthparts and the feeding behavior thereupon.     

Spectral, Physical, and Electron Transport Activities in the Photosynthetic Apparatus of Mesophyll Cells and Bundle Sheath Cells of Digitaria sanguinalis (L.) Scop

Isolated mesophyll cells and bundle sheath cells of Digitaria sanguinalis were used to study the light-absorbing pigments and electron transport reactions of a plant which possesses the C₄-dicarboxylic acid cycle of photosynthesis. Absorption spectra and chlorophyll determinations are presented showing that mesophyll cells have a chlorophyll a-b ratio of about 3.0 and bundle sheath cells have a chlorophyll a-b ratio of about 4.5. The absorption spectrum of bundle sheath cells has a greater absorption in the 700 nm region at liquid nitrogen temperature, and there is a relatively greater amount of a pigment absorbing at 670 nm in the bundle sheath cells compared to the mesophyll cells. Fluorescence emission spectra, at liquid nitrogen temperature, of mesophyll cells have a fluorescence 730 nm-685 nm ratio of about 0.82 and bundle sheath cells have a ratio of about 2.84. The reversible light-induced absorption change in the region of P₇₀₀ absorption is similar in both cell types but bundle sheath cells exhibit about twice as much total P₇₀₀ change as mesophyll cells on a total chlorophyll basis. The delayed light emission of bundle sheath cells is about one-half that of mesophyll cells. Both mesophyll cells and bundle sheath cells evolve oxygen in the presence of Hill oxidants with the mesophyll cells exhibiting about twice the activity of bundle sheath cells, and both activities are inhibited by 1 μM 3-(3,4-dichlorophenyl)-1, 1-dimethylurea. Ferredoxin nicotinamide adenine dinucleotide phosphate reductase is present in both cells although it is about 3- or 4-fold higher in mesophyll cells than in bundle sheath cells. Glyceraldehyde 3-P dehydrogenases, both nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, are equally distributed in the two cell types on a chlorophyll basis. Malic enzyme is localized in the bundle sheath cells.     

Morphology, anatomy and vasculature of leaves in Pinus (Pinaceae) and its evolutionary meaning

The morphology, anatomy and vasculature of Pinus leaves was studied. The results indicate that the Pinus leaf is always supplied with only one single vascular bundle, which is surrounded by a prominent bundle sheath. In several taxa, especially of subgenus Pinus, the vascular bundle may however be subdivided by longitudinal parenchymatic bands in the middle part of the leaf. As a result, the single bundle gets the appearance of two individual bundles, which are surrounded by a common bundle sheath. The general rule that a bundle sheath does sheath only one single bundle, as in other gymno- and angiospermous seed plants, applies therefore also for Pinus. The morphological and anatomical similarities between cladodes of Sciadopitys (Sciadopityaceae) and Pinus leaves are based on a completely different bauplan. The idea of “hidden cladodes” in some Pinaceae is therefore obsolete.     

Formation of Stylet Sheaths in āere (in air) from Eight Species of Phytophagous Hemipterans from Six Families (Suborders: Auchenorrhyncha and Sternorrhyncha)

Stylet sheath formation is a common feature among phytophagous hemipterans. These sheaths are considered essential to promote a successful feeding event. Stylet sheath compositions are largely unknown and their mode of solidification remains to be elucidated. This report demonstrates the formation and solidification of in āere (in air) produced stylet sheaths by six hemipteran families: Diaphorina citri (Psyllidae, Asian citrus psyllid), Aphis nerii (Aphididae, oleander/milkweed aphid), Toxoptera citricida (Aphididae, brown citrus aphid), Aphis gossypii (Aphididae, cotton melon aphid), Bemisia tabaci biotype B (Aleyrodidae, whitefly), Homalodisca vitripennis (Cicadellidae, glassy-winged sharpshooter), Ferrisia virgata (Pseudococcidae, striped mealybug), and Protopulvinaria pyriformis (Coccidae, pyriform scale). Examination of in āere produced stylet sheaths by confocal and scanning electron microscopy shows a common morphology of an initial flange laid down on the surface of the membrane followed by continuous hollow core structures with sequentially stacked hardened bulbous droplets. Single and multi-branched sheaths were common, whereas mealybug and scale insects typically produced multi-branched sheaths. Micrographs of the in āere formed flanges indicate flange sealing upon stylet bundle extraction in D. citri and the aphids, while the B. tabaci whitefly and H. vitripennis glassy-winged sharpshooter flanges remain unsealed. Structural similarity of in āere sheaths are apparent in stylet sheaths formed in planta, in artificial diets, or in water. The use of ‘Solvy’, a dissolvable membrane, for intact stylet sheath isolation is reported. These observations illustrate for the first time this mode of stylet sheath synthesis adding to the understanding of stylet sheath formation in phytophagous hemipterans and providing tools for future use in structural and compositional analysis.     

The developmental capacity of various stages of a macrocyst-forming strain of the cellular slime mold, Dictyostelium mucoroides.

Vegetative cells of certain strains of Dictyostelium mucoroides form fruiting bodies on an agar surface and macrocysts when placed under saline. This study sought to determine whether the aggregation and pseudoplasmodial stages of fruiting body formation could be induced to form macrocysts when placed under saline. Likewise, different stages in macrocyst formation were put on an agar surface to determine their potential to switch to fruiting body formation. It was found that 78% of the aggregates and 21% of the pseudoplasmodia placed under saline formed macrocysts indicating that as fruiting body development proceeds, there is a restriction of the capability of cells to respond to environmental conditions favoring macrocyst formation. Stages in macrocyst development prior to the formation of precysts always formed fruiting bodies when put on agar. Once precysts had formed, surrounded by their acellular sheath, they always developed as macrocysts on agar. Peripheral cells isolated from precysts and put on agar quickly aggregated; the aggregates became surounded by a sheath and developed as macrocysts. If isolated peripheral cells were allowed to proliferate on the agar surface, the resulting cells aggregated and formed fruiting bodies.     

A salivary sheath protein essential for the interaction of the brown planthopper with rice plants

Salivary secretions, including gel saliva and watery saliva, play crucial roles in the interaction between the insect and plant during feeding. In this study, we identified a salivary gland-specific gene encoding a salivary sheath protein (NlShp) in Nilaparvata lugens. NlShp has two alternative splicing variants; both are expressed at high levels during the nymph and adult stages. Immunohistochemical staining showed that the NlShp were synthesized in the principal gland cells of the salivary gland. LC-MS/MS and western blot analysis confirmed that NlShp was one of the components of the salivary sheath. Simultaneously knocking down the two NlShp variants by RNA interference inhibited both salivary flange and salivary sheath formation and resulted in a lethal phenotype within four days for the brown planthopper (BPH) feeding on rice plants, indicating that the salivary sheath and salivary flanges were essential for plant-associated feeding. Despite the salivary sheath deficiency, no obvious phenotype was observed in the NlShp-knockdown BPHs fed on artificial diet. The electrical penetration graph (EPG) results showed that salivary sheath-deficient BPHs exhibited a prolonged nonpenetration period, scarce sap period, and increased stylet movement on rice plants and eventually starved to death. Our results provided evidence that the interaction between the salivary sheath and host plant might be a critical step in successful BPH feeding. According to present research, we propose a salivary sheath required feeding model for piercing-sucking insects and provide a potential target for rice planthopper management.     

A col10a1:nlGFP transgenic line displays putative osteoblast precursors at the medaka notochordal sheath prior to mineralization

In teleosts, such as medaka, ossification of the vertebral column starts with the mineralization of the notochordal sheath in a segmental pattern. This establishes the chordal centrum, which serves as the basis for further ossifications by sclerotome derived osteoblasts generating the vertebral body. So far, it is unclear which cells produce the notochordal sheath and how a segmental pattern of mineralization is established in teleosts. Here, we use a transgenic medaka line that expresses nlGFP under the control of the col10a1 promoter for in vivo analysis of vertebral body formation. We show that col10a1:nlGFP expression recapitulates endogenous col10a1 expression. In the axial skeleton, col10a1:nlGFP cells appear prior to the mineralization of the notochordal sheath in a segmental pattern. These cells remain on the outer surface of the chordal centra during mineralization as well as subsequent perichordal ossification of the vertebral bodies. Using twist1a1:dsRed and osx:mCherry transgenic lines we show that a subset of col10a1:nlGFP cells is derived from sclerotomal precursors and differentiates into future osteoblasts. For the first time, this shows a segmental occurrence of putative osteoblast precursors in the vertebral centra prior to ossification of the notochordal sheath. This opens the possibility that sclerotome derived cells in teleosts are implicated in the establishment of the mineralized vertebral column in a similar manner as previously described for tetrapods.     

Electron Microscopy of Immune Disruption of Leptospires: Action of Complement and Lysozyme

Sequential disruption of the sheath of avirulent leptospires of the serotype canicola with antibody and complement was monitored by electron microscopy. Loosening and separation of the sheath from the protoplasmic cylinder was observed as early as 2 min after exposure to complement. Virulent leptospires of this serotype were morphologically intact after 1 hr of exposure to antibody and complement. Similarly, treatment of leptospires of the serotype patoc with normal serum and complement severely damaged the sheath structure. Removal of the sheath of both serotypes permitted lysozyme to act on the wall of the protoplasmic cylinder. Thus, morphological evidence for the location of the mucopeptide-containing structure of these leptospires was obtained. Viable leptospires with intact sheaths were resistant to lysozyme alone. Sections and negatively stained preparations of sheaths of serotypes canicola and patoc revealed three dense layers with two intermediate light zones and an overall thickness of about 110 A. A periodicity of 40 A was observed in sheath fragments produced by complement. The 70 A wallmembrane complex of leptospires of both serotypes consisted of two dense layers with an intermediate light zone. Structures apparent after removal of the outer sheath included membranous bodies or mesosomes, axial filaments attached to terminal knobs at opposite ends of the cell, and electron-dense intracellular bodies.