Transcuticular solute movement in parasitic nematodes: relationship between non-polar solute transport and partition coefficient

The rate constants (K) for steroid transport across the isolated cuticle of Ascaris suum were, linearly related to partition coefficients (P) of steroids in model solvents. The slope of log P vs log K for an isolated cuticle was closest to unity for the more polar solvents, octanol and ether, suggesting that the barrier to steroid movement across the cuticle has partition properties similar to these solvents. The transcuticular movement of steroids into intact adult A. suum and infective larvae of Haemonchus contortus was predicted by the P in model solvents suggesting that the same barriers for transcuticular solute movement may exist in intact worms as in isolated cuticles.     

Ultrastructure of the cell wall of Gracilaria cf. verrucosa (Gracilariales,Rhodophyta): effects of steam explosion

Ultrastructural (SEM, TEM) and cytochemical organization of the skeletal and matrix polysaccharides of fresh and steam exploded Gracilaria verrucosa thalli were compared to chemical analyses of the agar extracted after this treatment. Upon steam explosion, surface microfractures appeared in limited areas of the cortex. SEM preparations indicated that apical and medullary regions were the most affected, resulting in detachment of adjacent cells. In TEM sections, the cuticle of the surface layer exhibited deeply altered zones with a spongy appearance. In the cytoplasm starch grains were disrupted. The skeletal component of the immediate cell wall was maintained with a different organization of the microfibrils and was positive to periodic acid-thiosemicarbazide-silver proteinate. The intercellular matrix contained granules and fibrils and lacked a ruthenium red reaction. These results are consistent with lower sulfate-ester and higher glucose contents detected in agars extracted using steam explosion.     

Structure and function in the grass ligule: The membranous ligule ofLolium temulentum L. as a secretory organ

Summary Using phosphotungstic acid and periodic acid/thiocarbohydrazide/silver proteinate (Thiéry test) aspects of polysaccharide ultracytochemistry were studied in the membranous ligule ofLolium temulentum L. Staining results are presented for all three tissues-abaxial and adaxial epidermes and mesophyll-but discussed only for the epidermes. PTA- and PATAg-staining of the adaxial epidermis suggested synthesis of a conjugated polysaccharide material in this tissue, its accumulation in the periplasmic space and its subsequent secretion to the outside of the ligule via gaps in the cuticle. The ligule of this grass is considered to be a secretory organ.     

Cytochemical and ultrastructural studies of Candida albicans

Ultrastructural modifications of the cell wall coat of Candida albicans during adherence to host cells were investigated using various cytochemical techniques. Attachment of the fungus to buccal epithelial cells appeared to involve spatial rearrangement of their cell wall surface. In particular adhering yeast developed a fibrogranular surface layer visualized by the periodic acid — thiocarbohydrazide silver proteinate technique (a polysaccharide detectron technique); Concanavalin A binding sites detected on their cell wall coat were highly increased. Attachment of yeasts to epithelial cells appeared mediated by fibrillar structures or polysaccharidic granules distributed on the cell wall coat. But free extra-cell wall material containing mannoproteins released from the yeast surface suggested additional mechanisms.Abbreviations Con A Concanavalin A - Man-fer mannosyl ferritin - PATAg Periodic acid-thiocarbohydrazide-silver proteinate     

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

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

The Morphology and Permeability of Isolated Cuticular Membranes of Hoya carnosa R. Br. (Asclepiadaceae)

This investigation is in part an extension of previous leafcuticle observations made on 52 other taxa among 34 families.Dewaxed, chemically isolated, adaxial and abaxial cuticularmembranes and transverse leaf sections of the wax-flower plant(Hoya carnosa R. Br.) were examined using ordinary stainingtechniques and light-microscopy methods. Evidence is presentedfor the existence of ubiquitous, discrete, naturally occurringcuticular pores, concomitant with anticlinally oriented trans-cuticularcanals, distributed randomly throughout the cuticular matrix.The surface of the adaxial cuticular membrane contains approx.6540 unclustered pores per mm², the abaxial approx. 4680 poresper mm². Pore and canal diameters range between 0.5 and 0.75µm. The canals are often arcuate and their lengths aredirectly related to cuticle thickness. No correlations werefound between cuticle thickness and either pore numbers or poreand canal diameters. Based upon experiments with various pHindicators, solutions, and stains, the dewaxed, dry cuticularmembrane of H. carnosa appears to be both distinctly hydrophilicand selectively permeable through a myriad of microscopicallyvisible pores and canals permeating its matrix. A de novo interpretationof gross cuticle morphology based solely upon light microscopyobservations is presented by semi-diagrammatic illustrations. Hoya carnosa R. Br., wax-flower (wax-plant), cuticular membranes, cuticular pores, transcuticular canals, permeability     

CYTOPLASMIC MICROTUBULE AND WALL MICROFIBRIL ORIENTATION IN ROOT HAIRS OF RADISH

The fine structure of young root hairs of radish was studied, with special attention to cytoplasm-wall relationships. Hairs up to 130 µ in length were examined after fixation of root tips in glutaraldehyde followed by osmium tetroxide. Microtubules occur axially aligned in the cytoplasm just beneath the plasmalemma, and extend from the base of the hair to within 2 to 3 µ of the tip. Poststaining with uranyl acetate and lead citrate clearly reveals in thin sections the presence of the two layers of cellulose microfibrils known from studies on shadowed wall preparations: an outer layer of randomly arranged microfibrils arising at the tip, and a layer of axially oriented microfibrils deposited on the inside of this layer along the sides. The youngest microfibrils of the inner, oriented layer first appear at a distance of about 25 µ from the tip. Although the microfibrils of the inner layer and the adjacent microtubules are similarly oriented, the oriented microtubules also extend through the 20- to 25-µ zone near the tip where the wall structure consists of random microfibrils. This suggests that the role of microtubules in wall deposition or orientation may be indirect.     

Ultrastructure and development of the extensible abdominal intersegmental membranes of the female migratory locust

The cuticle of the retracted extensible membrane of the female locust is 300 mum thick and below its highly folded epicuticle there is a zone (5 mum thick) of helicoidally oriented laminae of microfibrils (lamellae), an elastomer layer (180 mum thick) of microfibrils with no preferred orientation and a subcuticular zone (1 mum thick). The epidermal cell layer has an extensive system of junctional specializations and pore canals traverse the cuticle to the helicoidally oriented lamellae. In the newly ecdysed adult the elastomer layer is absent and the helicoidally oriented lamellae are incomplete. Essentially the membrane consists of an elastomer layer contained between two stable layers cross-linked by pore canals, one to the other. When in the plasticized state the membrane combines low stiffness with high extensibility and during extension the elastomer layer flows. Recovery is effected by muscles and when the two stable layers have returned to their unstretched states the fluid elastomer is again evenly distributed. There is an increase in the water content and in the volume of the cuticle when it is fully extended. The ultrastructure of the extensible membrane is compared with those of the inextensible membranes from male and female locusts.     

Development of the crystalliferous cuticle of Chamaecyparis lawsoniana (A. Murr.) Pari. (Gupressaceae)

A developmental study of the cuticle has shown that it consists of a homogeneous cuticle proper apposed on the wall and a heterogeneous cuticular layer generated by intussusception of cutin into the wall. At an early stage, the adcrusted cuticle proper is underlain by a ruthenium red-positive layer in which the cuticular layer originates. The origin of the anticlinal flange is referable to an electron-dense, ruthenium red-positive ridge which arises above the anticlinal wall and which also becomes cutinized. At leaf maturity, the inner surface of the cuticular layer, including that of the flange, forms interdigitating protuberances with the cell wall.
Development of the cuticle coincides with deposition of crystals of calcium oxalate in the epidermal cell wall. Initiation of large, early-formed crystals is associated with electron-opaque membranous structures formed close and parallel to the plasmalemma in the young cell wall. Crystals undergo periclinal and anticlinal growth and subsequently become engulfed within the cuticle by development of the cuticular layer. Cutin/polysaccharide interaction during development and the significance of crystal deposition are discussed.     

Atomic force microscopy of cotton fiber cell wall surfaces in air and water: quantitative and qualitative aspects

Cotton (Gossypium hirsutum cv. MD51) fiber cell walls were analyzed with an atomic force microscope to determine the effect of chemical treatments on cell wall organization and topography. Analysis of fibers in either air or water and without any staining or coating produced high-resolution images of cell wall microstructure which could be used for detailed quantitative analysis. Treatment of fibers with 1% H₂O₂ had little effect on surface morphology. Alkali removed much of the cuticle, some primary wall components, and revealed mostly thin-diameter microfibrils. Acidic Updegraff reagent fragmented the fibers, removed much of the cuticle, and revealed mostly thick microfibrils. The surface roughness of fibers treated sequentially with alkali and acid was quantitatively distinguishable from all other fiber types based on the standard deviation of the height data, amplification of surface area, and integration of the scan line data. Analysis of the fractal dimension enabled untreated and peroxide-treated fibers to be clearly distinguished from the other fiber types. Segmentation of the fractal data revealed specific portions of the fractal dimension which were especially useful for defining the size of structures that differentiated fiber types. Areas containing microfibrils could be quantitatively differentiated from non-microfibrillar areas. In water, some alkali-treated fibers had microfibrils that were relatively small in diameter while others appeared to consist of crystalline arrays of smaller fibrils. Received: 10 December 1996 / Accepted: 29 January 1997     

Formation of root hairs by the wheat Triticum Vulgare Host. Trichoblasts.

The process of root hair formation has been studied by light-, transmission-and scanning electron microscopy. In the course of root hair development a break of the outer cell wall is observed by electron microscopy. It apparently occurs after the break of the fibrillar layer. The break of the outer layer of the cell wall is assumed to represent the break of the outer mucilage, the cuticle and the adjoining amorphous matrix with irregularly oriented cellulose microfibrils. The scheme of successive ultrastructural changes in the outer cell wall pattern during root hair formation is presented.     

Microtubules and morphogenesis in stomata of the water fernAzolla: An unusual mode of guard cell and pore development

Summary A mature stomate of the water fernAzolla consists of a single apparently unspecialized annular guard cell (GC) with two nuclei surrounding an elongated pore aligned longitudinally in the leaf. During development, the guard mother cell develops a preprophase band (PPB) of microtubules (MTs) oriented transverse to the leaf axis. This is followed by a cell plate which fuses with the parental walls at the PPB site. Subsequently only the central part of the cell plate is consolidated, while the parts to either side become perforated and tenuous and may disperse completely, forming a single composite GC.Meanwhile, a dense array of MTs appears along both faces of the central part of the new wall, oriented normal to the leaf surface. Further MT arrays radiate out across the periclinal walls from the region of the consolidated cell plate. Putative MT nucleating sites are seen along the cell edges between these anticlinal and periclinal arrays. Polarized light microscopy reveals cellulose deposition parallel to the periclinal MT arrays. At the same time lamellar material is deposited within the new anticlinal wall. As the GC complex elongates, a split appears in these lamellae creating an initially transverse slit which then opens up to become first circular and ultimately an elongated pore aligned in the long axis of the leaf,i.e., at right angles to the wall in which it originated. The radiating pattern of cellulose microfibrils in the periclinal walls contributes to the shaping of the pore. Elongation at the apical and basal ends of the GC is restricted by longitudinal microfibril orientation, while that at the sides is facilitated by transverse alignment.     

Apple Fruit Cuticles and the Occurrence of Pores and Transcuticular Canals

Developmental studies were made on the dewaxed thin-sectionedapple fruit cuticles of 10 Malus sylvestris Mill, cultivarsfrom 4 weeks before anthesis through fruit maturation and harvest.Cuticular development appears to correlate well with the generalgrowth of the fruit. However, no correlation exists betweenfruit size and cuticle thickness. Cuticular pores were evidentas early as 1 week before anthesis and transcuticular canalsbecame evident by 1 week following anthesis. Dewaxed thin-sectioned,as well as isolated, mature fruit cuticles of 16 cultivars andfour crab-apples consistently revealed the distinct presenceof ubiquitous pores and canals. Evidence is provided by lightmicroscopy and scanning electron microscopy (SEM). Measurementswere taken of cuticle thicknesses during development and ofcuticular pore dimensions, and calculations were made of poreand canal numbers. Fruit size alone is not directly indicativeof total pore numbers per surface area. Canal lengths are directlyrelated to the developmental thickness of the cuticle. No correlationwas found between the thickness of the mature cuticle and eitherthe number of pores present or the pore diameters. Malus sylvestris Mill, apple fruit, morphology, cuticle, cuticular flanges, cuticular pores, transcuticular canals, ultrastructure     

Changes in microfibril arrangement on the inner surface of the epidermal cell walls in the epicotyl of Vigna angularis ohwi et ohashi during cell growth

Throughout the entire period of cell growth, the microfibrils on the inner surface of the outer tangential walls of the epidermal cells of Vigna angularis epicotyls are running parallel to one another and their orientation differs from cell to cell. Although transverse, oblique and longitudinal microfibrils can be observed irrespective of cell age, the frequency distribution of microfibril orientation changes with age. In young cells, transversely oriented microfibrils predominate. In cells of medium age, which are still undergoing elongation, transverse, oblique and longitudinal microfibrils are present in quite similar frequencies. In old, non-growing cells, longitudinally oriented microfibrils are predominent. A decrease in the relative frequency of transversely oriented microfibrils with cell age was also observed in the radial epidermal walls.     

Deposition and reorientation of cellulose microfibrils in elongating cells of Petunia stylar tissue

According to Roelofsen and Houwink's (1953, Acta Bot. Neerl. 2, 218–225) multinet growth hypothesis, microfibrils originally deposited transversely in the cell wall become gradually reoriented towards more axial orientations during cell elongation. To establish the extent of reorientation, microfibrils were studied during their deposition and elongation, using stylar parenchyma and transmitting tissue cells of Petunia hybrida L. At the inner surface of very young cells, microfibrils were deposited in alternating Z- and S-helical orientations. The following sequence in deposition, from the exterior to the interior side of the wall, could be inferred: Axial: 150°–180° (Z-helical), 0°–30° (S-helical); oblique: 110°–150° (Z-helical), 30°–70° (S-helical); transverse: 90°–110° (Z-helical), 70°–90° (S-helical). With the increasing pitch, the density of the deposited microfibrils increased as well, giving rise to an alternating helical texture. During elongation, only transversely S- and Z-helically oriented microfibrils were deposited and all microfibrils underwent a certain reorientation as described in the multinet growth hypothesis. The texture resembled that of young cells and the wall maintained its thickness. The extent of passive reorientation was in agreement with the theoretical calculations made by Preston.Dedicated to Professor Dr. A.B. Wardrop, Melbourne, on the occasion of his 70th birthday     

Spatial relationship between microtubules and plasma-membrane rosettes during the deposition of primary wall microfibrils in Closterium sp.

The mechanism by which cortical microtubules (MTs) control the orientation of cellulose microfibril deposition in elongating plant cells was investigated in cells of the green alga, Closterium sp., preserved by ultrarapid freezing. Cellulose microfibrils deposited during formation of the primary cell wall are oriented circumferentially, parallel to cortical MTs underlying the plasma membrane. Some of the microfibrils curve away from the prevailing circumferential orientation but then return to it. Freeze-fracture electron microscopy shows short rows of particle rosettes on the P-face of the plasma membrane, also oriented perpendicular to the long axis of the cell. Previous studies of algae and higher plants have provided evidence that such rosettes are involved in the deposition of cellulose microfibrils. The position of the rosettes relative to the underlying MTs was visualized by deep etching, which caused much of the plasma membrane to collapse. Membrane supported by the MTs and small areas around the rosettes resisted collapse. The rosettes were found between, or adjacent to, MTs, not directly on top of them. Rows of rosettes were often at a slight angle to the MTs. Some evidence of a periodic structure connecting the MTs to the plasma membrane was apparent in freeze-etch micrographs. We propose that rosettes are not actively or directly guided by MTs, but instead move within membrane channels delimited by cortical MTs attached to the plasma membrane, propelled by forces derived from the polymerization and crystallization of cellulose microfibrils. More widely spaced MTs presumably allow greater lateral freedom of movement of the rosette complexes and result in a more meandering pattern of deposition of the cellulose fibrils in the cell wall.Abbreviations E-face exoplasmic fracture face - MT microtubule - P-face protoplasmic fracture-face     

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

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

Cell wall extension results in the coordinate separation of parallel microfibrils: evidence from scanning electron microscopy and atomic force microscopy

Enlargement of the cell wall requires separation of cellulose microfibrils, mediated by proteins such as expansin; according to the multi-net growth hypothesis, enlargement passively reorients microfibrils. However, at the molecular scale, little is known about the specific movement of microfibrils. To find out, we examined directly changes in microfibril orientation when walls were extended slowly in vitro under constant load (creep). Frozen-thawed cucumber hypocotyl segments were strained by 20-30% by incubation in pH 4.5 buffer or by incubation of heat-inactivated segments in alpha-expansin or a fungal endoglucanase (Cel12A). Subsequently, the innermost layer of the cell wall was imaged, with neither extraction nor homogenization, by field-emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM). AFM images revealed that sample preparation for FESEM did not appreciably alter cell wall ultrastructure. In both FESEM and AFM, images from extended and non-extended samples appeared indistinguishable. To quantify orientational order, we used a novel algorithm to characterize the fast Fourier transform of the image as a function of spatial frequency. For both FESEM and AFM images, the transforms of non-extended samples were indistinguishable from those of samples extended by alpha-expansin or Cel12A, as were AFM images of samples extended by acidic buffer. We conclude that cell walls in vitro can extend slowly by a creep mechanism without passive reorientation of innermost microfibrils, implying that wall loosening agents act selectively on the cross-linking polymers between parallel microfibrils, rather than more generally on the wall matrix.     

Peltate trichomes in the dormant shoot apex of Metrodorea nigra,a Rutaceae species with rhythmic growth

• In Metrodorea nigra, a Rutaceae species with rhythmic growth, the shoot apex in the dormant stage is enclosed by modified stipules. The young organs are fully covered with peltate secretory trichomes, and these structures remain immersed in a hyaline exudate within a hood-shaped structure. Our study focused on the morpho-functional characterization of the peltate trichomes and cytological events associated with secretion.
• Shoot apices were collected during both dormant and active stages and processed for anatomical, cytochemical and ultrastructural studies.
• Trichomes initiate secretion early on, remain active throughout leaf development, but collapse as the leaves expand; at which time secretory cavities start differentiation in the mesophyll and secretion increases as the leaf reaches full expansion. The subcellular apparatus of the trichome head cells is consistent with hydrophilic and lipophilic secretion. Secretion involves two vesicle types: the smaller vesicles are PATAg-positive (periodic acid/thiocarbohydrazide/silver proteinate) for carbohydrates and the larger ones are PATAg-negative. In the first phase of secretory activity, the vesicles containing polysaccharides discharge their contents through exocytosis with the secretion accumulating beneath the cuticle, which detaches from the cell wall. Later, a massive discharge of lipophilic substances (lipids and terpenes/phenols) results in their accumulation between the wall and cuticle. Release of the secretions occurs throughout the cuticular microchannels.
• Continued protection of the leaves throughout shoot development is ensured by replacement of the collapsed secretory trichomes by oil-secreting cavities. Our findings provide new perspectives for understanding secretion regulation in shoot apices of woody species with rhythmic growth.