COMPARATIVE STUDIES ON THE CELL WALLS OF SEXUAL AND ASEXUAL BANGIA ATROPURPUREA (RHODOPHYTA). II ELECTROPHORETIC PATTERNS OF POLYSACCHARIDES1

Cellulose acetate electrophoresis of the hot water soluble polysaccharide extracts from whole filaments, as well as base, mid and tip segments, of marine asexual and sexual Bangia atropurpurea (Roth) C. Ag. Yielded distinctive patterns which demonstrated that changes occur in the outer cell walls during sexual reproduction. Heterogeneity of the sulfated polysaccharide components isolated from outer cell walls was shown to be specifically related to sexual reproduction. Two components (Band I and II) were detected in extracts from tips of sexual filaments, whole only one (Band I) was present in the vegetative segments of all filaments and in asexual reproductive regions. The faster running component (Band II) was detected during the later stages of sexual development, prior to maturation.     

Micro-heterogeneity of pectins and calcium distribution in the epidermal and cortical parenchyma cell walls of flax hypocotyl

Summary Pectic polysaccharides are major components of the plant cell wall matrix and are known to perform many important functions for the plant. In the course of our studies on the putative role of pectic polysaccharides in the control of cell elongation, we have examined the distribution of polygalacturonans in the epidermal and cortical parenchyma cell walls of flax seedling hypocotyls. Pectic components have been detected with (1) the nickel (Ni²⁺) staining method to visualize polygalacturonates, (2) monoclonal antibodies specific to low (JIM5) and highly methylesterified (JIM7) pectins and (3) a combination of subtractive treatment and PATAg (periodic acid-thiocarbohydrazide-silver proteinate) staining. In parallel, calcium (Ca²⁺) distribution has been imaged using SIMS microscopy (secondary ion mass spectrometry) on cryo-prepared samples and TEM (transmission electron microscopy) after precipitation of calcium with potassium pyroantimonate. Our results show that, at the tissular level, polygalacturonans are mainly located in the epidermal cell walls, as revealed by the Ni²⁺ staining and immunofluorescence microscopy with JIM5 and JIM7 antibodies. In parallel, Ca²⁺ distribution points to a higher content of this cation in the epidermal walls compared to cortical parenchyma walls. At the ultrastructural level, immunogold labeling with JIM5 and JIM7 antibodies shows a differential distribution of pectic polysaccharides within cell walls of both tissues. The acidic polygalacturonans (recognized by JIM5) held through calcium bridges are mainly found in the outer part of the external wall of epidermal cells. In contrast, the labeling of methylesterified pectins with JIM7 is slightly higher in the inner part than in the outer part of the wall. In the cortical parenchyma cells, acidic pectins are restricted to the cell junctions and the wall areas in contact with the air-spaces, whereas methylesterified pectins are evenly distributed all over the wall. In addition, the pyroantimonate precipitation method reveals a clear difference in the Ca²⁺ distribution in the epidermal wall, suggesting that this cation is more tightly bound to acidic pectins in the outer part than in the inner part of that wall. Our findings show that the distribution of pectic polysaccharides and the nature of their linkages differ not only between tissues, but also within a single wall of a given cell in flax hypocotyls. The differential distribution of pectins and Ca²⁺ in the external epidermal wall suggests a specific control of the demethylation of pectins and a central role for Ca²⁺ in this regulation.Abbreviations Cdta diamino-1,2-cyclohexane tetra-acetic acid - PATAg periodic acid-thiocarbohydrazide-silver proteinate - PGA polygalacturonic acid - PME pectin methylesterase - RG I rhamnogalacturonan I - SIMS secondary ion mass spectrometry - TEM transmission electron microscopy     

A Histochemical Study of Cytoplasmic Changes During Wall Layer Formation in the Oospore of Albugo candida

The young multinucleate oogonium in Albugo is double-walled with an outer layer exhibiting a negative staining reaction for insoluble polysaccharides and an inner layer which is strongly PAS-positive. The oogonial nuclei exhibit an unusual staining behaviour with aniline blue showing an outer dark blue sheath of proteins surrounding a central hyaline nuclear core. Various histochemical localizations were performed for tracing the chronological sequence of development of the wall layers of the oospore. The first wall of the fertilized oospore was laid at the interphase of the periplasm and the ooplasm. Subsequent wall layers were formed both on the inner and outer side of the first oosporic wall. The second oosporic wall was formed just internal to the first one and exhibited faint PAS positivity. The third wall of the oospore was formed external to the first one and the PAS-negative material for this was apparently contributed by the periplasm. This wall layer at later stages acquired a ridged appearance and these ridges in a mature oospore appear as distinct “pegs”. The last wall to be formed is the innermost one and it completely surrounds the central ooplasm. This wall layer is callosic in nature.     

The ultrastructure of the cell wall of normal and apolar embryos ofFucus vesiculosus

Summary Structure and composition of the walls of normal and apolar embryos ofFucus vesiculosus L. were studied. Fucoidin was found in an amorphous outer layer and in an inner fibrillar layer of the wall, mainly at the rhizoid pole. Also in apolar embryos this inner layer was present; it was markedly thickened at the presumptive site of rhizoid formation.We suggest that initiation and extension of the rhizoid is accompanied by apposition of new fibrillar wall material containing sulphated polysaccharides on the inner side of the wall at the rhizoid pole. In apolar embryos this material accumulates at this pole.     

THE ULTRASTRUCTURE OF PHYCOPELTIS (CHROOLEPIDACEAE: CHLOROPHYTA). I. SPOROPOLLENIN IN THE CELL WALLS

Electron microscope observations on Phycopeltis epiphyton, a subaerial green alga found growing on the leaves of vascular plants and bryophytes, revealed the presence of a densely staining material within the inner and outer zones of the cell walls. This material resists acetolysis, is degraded by chromic acid, is unaffected by ethanolamine and exhibits secondary fluorescence when stained with the fluorochrome Primuline. These characteristics, together with infrared absorption spectra indicate that, on the basis of currently accepted criteria, the densely staining material is a sporopollenin and that it is a major component of the cell wall. Tests for cellulose, chitin, and lignin were negative, and little if any silica is present. It is suggested that negative results in tests for cellulose may be due to a masking effect by the sporopollenin. Comparison of the fine structure of the cell walls of P. epiphyton, pollen grains, and algal cells (known to contain sporopollenin) supports the suggestion that sporopollenin deposition on “unit membranes” is universal. Morphological similarity among sporopollenin lamellae in P. epiphyton, pollen grains, spores of land plants, and the trilaminar sporopollenin sheath in Chlorella, Scenedesmus, and Pediastrum indicates that the structures may be analogous. As in pollen grains, sporopollenin may provide protection against desiccation and parasitism. It may also be involved in the adhesion of Phycopeltis to host plants and in the adhesion between adjacent filaments of the thallus.     

Electron microscope studies on Leucothrix mucor

Summary Electron microscopic studies of thin sections of filaments, knots, resettes, gonidia, and gonidial-forming filaments of Leucothrix mucor were carried out. The cell wall is typical of gram-negative bacteria, with a double outer layer of variable thickness, a single thin middle layer which is probably peptidoglycan, and a double inner layer which is the cell membrane. The transverse septa of these filaments show two peptidoglycan layers, and no clearly demarked outer layer. During gonidial formation, there is a gradual rounding up of the cells, and the transverse septa become part of the gonidial wall. The cell membrane contains many invaginations, both along the outer wall and along the transverse septa. Thin sections through rosettes show the holdfast as material which is a heavily-staining amorphous material peripheral to the outer wall layer. Sections through knots show highly contorted cell walls, closely appressed. Fibrillar nuclear material, ribosomes, and storage granules can be seen within the cytoplasmic matrix.     

Lysis of cell walls ofMucor ramannianus möller by aStreptomyces sp.

A study has been made of some chemical and ultrastructural changes that occur in the hyphal, arthrospore and sporangiospore walls ofMucor ramannianus during lysis by a soil streptomycete.Arthrospore and hyphal walls, which were shown to contain chitin, chitosan, other polysaccharides and phosphate (principally as polyphosphate), were lysed by culture fluid of the streptomycete after this organism had been grown on the same material. Alcohol-insoluble material found in the supernatants of the incubation mixtures gave on hydrolysis glucosamine, galactose, mannose and fucose. No laminarinase activity was detected in these culture fluids. Culture fluids of the streptomycete after growth on chitin and chitosan were also found to lyse the walls of arthrospores and hyphae.Despite the chemical similarities the walls were very different in thin section.A major component in the sporangiospore walls was glucan and an active laminarinase was shown to be present in the culture fluids of the streptomycete after growth on them. Further, ultrathin sections showed that an inner fibrillar layer of the sporangiospore wall was lysed leaving an outer electron-dense layer.     

Turbidity affects association behaviour in male Poecilia latipinna

Male association preferences in a bisexual‐unisexual species complex were studied in clear and turbid environments. In south and central Texas, where the gynogenetic sexual‐parasite Poecilia formosa lives syntopically with Poecilia latipinna as its sexual host species, association times of P. latipinna males with conspecific sexual and heterospecific asexual females in clear and turbid water were measured sequentially. Turbidity had an influence on male mate association behaviour. Males spent less time with any kind of female stimulus in turbid water. There was no preference for conspecific sexual females, either in turbid water or under clear conditions. Also, origin of males and acclimatization to turbid water had no effect. How turbidity as a source of visual noise might affect communication among individuals and how this environmental factor might contribute to the stability of this sexual‐asexual mating complex in nature are discussed.     

Cell wall functions in growth and development —a physical and chemical point of view

The plant cell changes its cell wall architecture during growth and development through synthesis and degradation of wall polysaccharides. Changes of chemical components in the cell wall include not only the synthesis and degradation but also the shift of molecular-weight distribution of certain species of the component polysaccharides. The changes in chemical structure, in turn lead to alteration of physical properties of the cell wall. Changes of physical parameters of cell walls obtained by a physical method accord with the biochemical degradation of polysaccharides. The changes in chemical structures of the cell wall are regulated by plant hormones, stress signals and gene expression. The physical and chemical studies of the cell wall have disclosed that degradation and/or depolymerization of wall polysaccahrides causes decrease in viscosity of the cell wall, leading further extension of the cell wall even under the unchanged osmotic relation. Furthermore, cell walls of outer and inner tissues play different regulatory roles in tissue growth and stem strength was governed by the number of cellulose molecules in the cell wall. Recipient of the Botanical Society Award for Young Scientists, 1990.     

Regulation of pectic polysaccharide domains in relation to cell development and cell properties in the pea testa

The occurrence of pectic polysaccharide epitopes in cells and tissues of the pea testa during late stages of seed development have been examined in relation to anatomy and cell properties. Homogalacturonan, in a highly methyl-esterified form, was present throughout late development in all pea testa cell walls, including the thickened cell walls of the outer macrosclereid layer. Two epitopes, characteristic of the side-chains of the rhamnogalacturonan-I domain of pectic polysaccharides, occurred in restricted and separate cell layers of the pea testa. A (1-->4)-beta-D-galactan epitope was restricted to regions of the outer cell wall of the testa and to inner regions of the macrosclereid layer at 20 DAA and was absent from the osteosclereid and parenchyma cell walls. By 25 DAA the (1-->4)-beta-D-galactan epitope occurred only in the outer epidermal cell walls. A (1-->5)-alpha-L-arabinan epitope was also dependent on the developmental stage of the seed and was found with greatest abundance in the walls of the inner parenchyma cells. Cell separation studies indicated that, although calcium cross-links were involved in the maintenance of the link between the macrosclereid layer and proximal cell layers, most cell-to-cell adhesion in the testa was not due to calcium- or ester-based bonds.     

Effects of Lysozyme and Inorganic Anions on the Morphology of Streptococcus mutans BHT: Electron Microscopic Examination

The effects of hen egg white lysozyme and the inorganic salt sodium thiocyanate on the integrity of Streptococcus mutans BHT were studied by transmission electron microscopy. Both control cells and cells exposed to NaSCN possessed thick outer cell walls and densely staining inner cell walls juxtaposed to the plasma membranes. In the presence of NaSCN, however, the S. mutans BHT nucleoid was coagulated into thick electron-dense filaments. Exposure of S. mutans BHT to 150 μg of hen egg white lysozyme per ml resulted in the progressive destruction of both the cell walls and the plasma membranes. The enzyme appeared to affect the region of the cell wall septum, and exposure to 150 μg of hen egg white lysozyme per ml for as short a time as 10 min resulted in visible morphological cell wall alterations. At 30 min, ultrastructural observations revealed that the majority of the cells were in the process of expelling a portion of their cytoplasmic contents from the septal and other regions of the cells at the time of fixation. After 3 h of incubation in the presence of this high lysozyme concentration, gelled protoplasmic masses, which were free from the cells, were evident. In addition, extensive damage to the outer and inner cell walls and to the plasma membranes was apparent, although the cells maintained their shape. On some areas of the cell surface, the outer cell wall and plasma membrane were completely absent, whereas at other locations the outer cell wall was either split away from the inner cell wall and plasma membrane or distended from an area free of inner cell wall and plasma membrane. Upon addition of NaSCN to the hen egg white lysozyme-treated cells, both the gelled protoplasmic masses and the damaged cells exhibited an exploded appearance and existed as membrane ghosts, cell wall fragments, or dense aggregates of cytoplasmic components. The effects of a low lysozyme concentration (22.5 μg/ml) on S. mutans morphology were less pronounced at short incubation times (i.e., 10 and 30 min) than those that were observed with a high enzyme concentration; however, breaks in the cell walls and dissolution of the plasma membranes with resulting cell lysis were visible after a prolonged (3-h) incubation and after subsequent addition of NaSCN.     

Polarized microtubule deposition coincides with wall ingrowth formation in transfer cells ofVicia faba L. cotyledons

Summary The epidermal transfer cells in developingVicia faba L. cotyledons are highly polarized. Extensive wall ingrowths occur on their outer periclinal walls and extend part way down both anticlinal walls. This ingrowth development serves to increase the surface area of the plasma membrane and thus maximize porter-dependent uptake of sugars from the seed apoplasm. In contrast, the inner periclinal walls of these transfer cells do not form wall ingrowths. We have commenced a study of the mechanisms responsible for establishing this polarity by first analysing the microtubule (MT) cytoskeleton in developing transfer cells. Thin sections of fixed cotyledons embedded in methacrylate resin were processed for immunofluorescence microscopy using monoclonal anti--tubulin and counterstained with Calcofluor White to visualize wall ingrowths. In epidermal cells of young cotyledons where wall ingrowths were yet to develop, MT labelling was detected around all cortical regions of the cell. However, in cells where wall ingrowths were clearly established, MT labelling was detected almost exclusively in cortical regions adjacent to the wall ingrowths. Little, if any, MT labelling was detected on the anticlinal or inner periclinal walls of these cells. This distribution of MTs was most prominent in cells with well developed wall ingrowths. In these cells, a subpopulation of MTs were also detected emanating from the subcortex and extending towards the wall ingrowth region. The possible role of MT distribution in establishing transfer cell polarity and wall ingrowth formation is discussed.Abbreviations MT microtubule     

Spatial regulation of pectic polysaccharides in relation to pit fields in cell walls of tomato fruit pericarp

Scanning electron microscopic examination of intact tomato (Lycopersicon esculentum) pericarp and isolated pericarp cell walls revealed pit fields and associated radiating ridges on the inner face of cell walls. In regions of the cell wall away from pit fields, equivalent ridges occurred in parallel arrays. Treatment of isolated cell walls with a calcium chelator resulted in the loss of these ridges, indicating that they contain homogalacturonan-rich pectic polysaccharides. Immunolabeling procedures confirmed that pit fields and associated radiating ridges contained homogalacturonan. Epitopes of the side chains of pectic polysaccharides were not located in the same regions as homogalacturonan and were spatially regulated in relation to pit fields. A (1-->4)-beta-galactan epitope was absent from cell walls in regions of pit fields. A (1-->5)-alpha-arabinan epitope occurred most abundantly at the inner face of cell walls in regions surrounding the pit fields.     

ZYGOSPORE GERMINATION IN CHLAMYDOMONAS MONOICA (CHLOROPHYTA): TIMING AND PATTERN OF SECONDARY ZYGOSPORE WALL DEGRADATION IN RELATION TO CYTOPLASMIC EVENTS

The Chlamydomonas monoica Strehlow zygospore is a dormant heavily walled spore adapted to survive extreme environmental conditions. The zygospore wall is multilayered and includes an acetolysis-resistant component related to the general class of compounds referred to as sporopollenin. Germination of the zygospore requires induction and completion of nuclear meiotic divisions, cytokineses to produce the four vegetative progeny cells, and breakdown of the zygospore wall to allow progeny release. Analysis of zygospore wall breakdown by transmission electron microscopy of synchronously germinating zygospores revealed differences in the timing and nature of disintegration of the various wall layers. Breakdown of the outer trilamellar sheath occurred within 6 h after light induction, concomitant with the onset of prophase I. At the same time, stored lipid bodies were consumed and replaced by large cytoplasmic vacuoles. Degradation of the inner more massive wall layer was initiated several hours later at about the time of the second meiotic division. In areas beneath breaks in the trilamellar sheath, a fibrous electron opaque bridge of wall material was retained whereas degradation of the remainder of the inner layer progressed. Finally, disintegration of this bridge material, after the completion of the meiotic divisions and synthesis of progeny cell walls, resulted in the opening of large slits in the trilamellar sheath, allowing escape of the flagellated vegetative progeny. The chloroform resistance typical of mature zygospores was lost at approximately the same time that the initial breaks in the trilamellar sheath were detected but before disintegration of the inner wall layer(s).     

ULTRASTRUCTURAL OBSERVATIONS ON THE TRICHOBLASTS OF EQUISETUM

Equisetum trichoblasts are densely cytoplasmic, containing numerous starch-containing plastids, mitochondria, and concentrations of rough endoplasmic reticulum with attached polysomes. Numerous vesicles of Golgi origin are present, containing a lightly staining fibrillar material; these vesicles appear to fuse with the wall. The outer tangential and radial walls become thickened while the inner tangential wall remains thin with numerous plasmodesmata. As the trichoblasts develop into root hairs, vacuolation occurs, resulting in large vacuoles. This may represent autolytic vacuolation. The cytoplasm of the root hairs is similar to that of the trichoblasts.     

小麦条锈菌吸器超微结构和细胞化学的研究

本文应用电镜技术和细胞化学方法,对小麦条锈菌吸器和入侵点的超微结构进行了研究。小麦条锈菌吸器由呈管状的颈部和顶端膨大的吸器体组成,颈部壁和吸器体壁相互连贯,均为两层,并且含有多糖物质。在颈部中段存在有一染色较深的颈环结构。观察发现吸器中的多核现象极为普遍。细胞化学染色结果表明:在吸器外间质内分布有多糖物质;经蛋白消酶解处理后,吸器外间质中可观察到染色较深的纤丝状物质。在入侵点部位,吸器母细胞壁因局部增厚而呈凸镜状,入侵栓壁由内、外两层构成,这两层分别与吸器母细胞壁的第六层和第五层相连接。本研究还观察到同一入侵点产生两个入侵栓的现象。     

Cell wall polysaccharide distribution in Sandersonia aurantiaca flowers using immuno-detection

The localization of cell wall polysaccharides of the fused petals of monocotyledonous Sandersonia aurantiaca flowers has been identified using antibodies directed to pectin and xyloglucan epitopes and detection by fluorescence microscopy. Cross sections of the petal tissue were taken from cut flowers in bud and at various stages of maturity and senescence. Patterns of esterification in pectin backbones were identified by JIM5 and 2F4 labelling. Pectic galactan and arabinan side branches were detected by LM5 and LM6, respectively, while fucosylated xyloglucan was identified by CCRC-M1. The labelling patterns highlighted compositional differences between walls of the outer/inner epidermis compared to the spongy parenchyma cells of the interior mesophyll for fucosylated xyloglucan and arabinan. Partially esterified homogalacturonan was present in the junction zones of the outer epidermis and points of contact between cells of the mesophyll, and persisted throughout senescence. Pectic galactans were ubiquitous in the outer and inner epidermal cell walls and walls of the interior mesophyll at flower opening, whereas pectic arabinan was found predominantly in the epidermal cells. Galactan was lost from walls of all cells as flowers began to senesce, while fucosylated xyloglucan appeared to increase over this time. Such differences in the location of polysaccharides and the timing of changes suggest distinct combinations of certain polysaccharides offer mechanical and rheological advantages that may assist with flower opening and senescence.     

FINE STRUCTURE OF THE GRAM-NEGATIVE BACTERIUM ACETOBACTER SUBOXYDANS

The morphological features of the cell wall, plasma membrane, protoplasmic constituents, and flagella of Acetobacter suboxydans (ATCC 621) were studied by thin sectioning and negative staining. Thin sections of the cell wall demonstrate an outer membrane and an inner, more homogeneous layer. These observations are consistent with those of isolated, gram-negative cell-wall ghosts and the chemical analyses of gram-negative cell walls. Certain functional attributes of the cell-wall inner layer and the structural comparisons of gram-negative and gram-positive cell walls are considered. The plasma membrane is similar in appearance to the membrane of the cell wall and is occasionally found to be folded into the cytoplasm. Certain features of the protoplasm are described and discussed, including the diffuse states of the chromatinic material that appear to be correlated with the length of the cell and a polar differentiation in the area of expected flagellar attachment. Although the flagella appear hollow in thin sections, negative staining of isolated flagella does not substantiate this finding. Severe physical treatment occasionally produces a localized penetration into the central region of the flagellum, the diameter of which is much smaller then that expected from sections. A possible explanation of this apparent discrepancy is discussed.     

Spectroscopic and biochemical analysis of regions of the cell wall of the unicellular 'mannan weed', Acetabularia acetabulum

Although the Dasycladalean alga Acetabularia acetabulum has long been known to contain mannan-rich walls, it is not known to what extent wall composition varies as a function of the elaborate cellular differentiation of this cell, nor has it been determined what other polysaccharides accompany the mannans. Cell walls were prepared from rhizoids, stalks, hairs, hair scars, apical septa, gametophores and gametangia, subjected to nuclear magnetic resonance and Fourier transform infrared spectroscopy, and analyzed for monosaccharide composition and linkage, although material limitations prevented some cell regions from being analyzed by some of the methods. In diplophase, walls contain a para-crystalline mannan, with other polysaccharides accounting for 10-20% of the wall mass; in haplophase, gametangia have a cellulosic wall, with mannans and other polymers representing about a quarter of the mass. In the walls of the diplophase, the mannan appears less crystalline than typical of cellulose. The walls of both diploid and haploid phases contain little if any xyloglucan or pectic polysaccharides, but appear to contain small amounts of a homorhamnan, galactomannans and glucogalactomannans, and branched xylans. These ancillary polysaccharides are approximately as abundant in the cellulose-rich gametangia as in the mannan-rich diplophase. In the diplophase, different regions of the cell differ modestly but reproducibly in the composition of the cell wall. These results suggest unique cell wall architecture for the mannan-rich cell walls of the Dasycladales.     

Differential Effect of Auxin on Molecular Weight Distributions of Xyloglucans in Cell Walls of Outer and Inner Tissues from Segments of Dark Grown Squash (Cucurbita maxima Duch.) Hypocotyls

Effects of indole-3-acetic acid (IAA) on the mechanical properties of cell walls and structures of cell wall polysaccharides in outer and inner tissues of segments of dark grown squash (Cucurbita maxima Duch.) hypocotyls were investigated. IAA induced the elongation of unpeeled, intact segments, but had no effect on the elongation of peeled segments. IAA induced the cell wall loosening in outer tissues as studied by the stress-relaxation analysis but not in inner tissues. IAA-induced changes in the net sugar content of cell wall fractions in outer and inner tissues were very small. Extracted hemicellulosic xyloglucans derived from outer tissues had a molecular weight about two times as large as in inner tissues, and the molecular weight of xyloglucans in both outer and inner tissues decreased during incubation. IAA substantially accelerated the depolymerization of xyloglucans in outer tissues, while it prevented that in inner tissues. These results suggest that IAA-induced growth in intact segments is due to the cell wall loosening in outer tissues, and that IAA-accelerated depolymerization of hemicellulosic xyloglucans in outer tissues is involved in the cell wall loosening processes.