Volvocine cell walls and their constituent glycoproteins: An evolutionary perspective

Summary Similarities in the composition of the extracellular matrix suggest that only some species of the unicellularChlamydomonas are closely related to the colonial and multicellular flagellated members of the family Volvocaceae. The cell walls from all of the algae in this volvocine group contain a crystalline layer. This lattice structure can be used as a phylogenetic marker to divideChlamydomonas species into distinct classes, only one of which includes the volvocacean algae. Similarly, not all species ofChlamydomonas are sensitive to each other's cell wall lytic enzymes, implying divergence of the enzyme's inner wall substrate. Interspecific reconstitution of the crystalline layer is possible betweenC. reinhardtii and the multicellularVolvox carteri, but not betweenC. reinhardtii andC. eugametos. The hydroxyproline-rich glycoproteins (HRGPs) which make up the crystalline layer in genera which have a similar crystal structure exhibit many homologies. Interestingly, the evolutionarily distant cell walls ofC. reinhardtii andC. eugametos also contain some HRGPs displaying a few morphological and amino acid sequence homologies. The morphological similarities between the flagellar agglutinins (HRGPs responsible for sexual recognition and adhesion during the mating reaction) and the cell wall HRGPs leads to the proposal of a superfamily from which novel HRGPs (designed for self-assembly/recognition) can constantly evolve. Just as variations in the wall HRGPs can lead to unique wall structures, new agglutinins facilitate sexual isolation of new species. Thus, the HRGPs could emerge as valuable phylogenetic markers.Abbreviations GLE gametic lytic enzyme - GP glycoprotein - HRGP hydroxyproline-rich glycoprotein - SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - VLE vegetative lytic enzyme - VSP vegetative serine/proline-rich - WP wall protein - ZSP zygotic serine/proline-rich     

CELL WALL CHEMISTRY AND ULTRASTRUCTURE OF CHLOROCOCCUM OLEOFACIENS (CHLOROPHYCEAE)1,2

Cell walls of Chlorococcum oleofadens Trainor & Bold were examined ultrastructurally and chemically. The wall of zoospores has a uniform 30 nm width and a regular lamellar pattern. Zoospores and young vegetative cell walk exhibit periodicities, consisting of 20 nm ridges on the outer layer. Vegetative cell walls have a variable thickness of Up to 800 nm and are composed of multiple layers of electron dense material. Further, vegetative walk contain a microfibrillar material composed predominantly of glucose and presumed to be cellulose. Except for this cellulose, vegetative cell wall chemistry is very similar to that of Chlamydomemas being composed of glycoprotein rich in hydroxyproline. The hydroxyproline in Chlorococcum walls is linked glycosidically to a mixture of hetrooligosaccharides composed of arabinose and galactose, and in one instance, an unknown 6-deoxyhexose. Altogether, the glycoprotein complex accounts for at least 52% of the wall. The amino acid composition of the walls is stikingly similar to those of widely different plant species. Indirect evidence indicates zoospore cell walls are also chemically similar to those of Chlamydomonas, and like them, are cellulose free. Thus a major chemical difference between zoospore and vegetative cell walk of Chlorococcum is the presence of cellulose in the latter. The contribution of this microfibrillar cellulose to the physical properties of the vegetative wall is discussed.     

Crystalline order to high resolution in the sheath of Methanospirillum hungatei: a cross-beta structure

Electron microscopy and electron diffraction indicate that the outer sheath of the cell wall of the archaebacterium Methanospirillum hungatei contains a two-dimensional crystalline lattice having, at least to low resolution, p2 symmetry in projection with a = 5.66 nm, b = 2.81 nm and gamma = 85.6 degrees. At a resolution of 2 nm, the unit cell contains two lobes, whereas high-angle electron diffraction shows the presence of a substantial quantity of beta structure, with the 0.47 nm spacing (between polypeptide chains within a sheet) oriented circumferentially. The sheath is unusual when compared to other regular surface arrays found on bacteria in that it is a compact structure with small subunits. It may have a structural role analogous to barrel hoops since it tends to fragment perpendicular to its axis to give rings or hoops.     

Three-dimensional structure of a cell wall glycoprotein

Electron microscopy and computer image analysis have been used to determine the three-dimensional structure of the crystalline glycoprotein cell wall layer of the alga Lobomonas piriformis. Images of negatively stained specimens, tilted through a range of angles up to 70 °, were combined to give a map of the molecular envelope to a resolution of 2.0 nm. The cell wall layer consists of crystalline plates the centres and edges of which display distinctly different but isomorphous structures. A comparison of three-dimensional reconstructions of the two areas shows the difference probably to be due to a conformational change of one of the glycoprotein subunits. The structure consists of two sets of dimers composed of rod-shaped subunits which lie with their long axes approximately in the plane of the crystal. The centre-edge transition may have significance in the pathway of accretion of new subunits during cell wall growth.     

CELL WALL CARBOHYDRATE EPITOPES IN THE GREEN ALGA OEDOGONIUM BHARUCHAE F. MINOR (OEDOGONIALES,CHLOROPHYTA)1

Cell wall changes in vegetative and suffultory cells (SCs) and in oogonial structures from Oedogonium bharuchae N. D. Kamat f. minor Vélez were characterized using monoclonal antibodies against several carbohydrate epitopes. Vegetative cells and SCs develop only a primary cell wall (PCW), whereas mature oogonial cells secrete a second wall, the oogonium cell wall (OCW). Based on histochemical and immunolabeling results, (1→4)‐β‐glucans in the form of crystalline cellulose together with a variable degree of Me‐esterified homogalacturonans (HGs) and hydroxyproline‐rich glycoprotein (HRGP) epitopes were detected in the PCW. The OCW showed arabinosides of the extensin type and low levels of arabinogalactan‐protein (AGP) glycans but lacked cellulose, at least in its crystalline form. Surprisingly, strong colabeling in the cytoplasm of mature oogonia cells with three different antibodies (LM‐5, LM‐6, and CCRC‐M2) was found, suggesting the presence of rhamnogalacturonan I (RG‐I)–like structures. Our results are discussed relating the possible functions of these cell wall epitopes with polysaccharides and O‐glycoproteins during oogonium differentiation. This study represents the first attempt to characterize these two types of cell walls in O. bharuchae, comparing their similarities and differences with those from other green algae and land plants. This work represents a contribution to the understanding of how cell walls have evolved from simple few‐celled to complex multicelled organisms.     

Crystalline fibril structure of type II collagen in lamprey notochord sheath

We report here the existence of a crystalline molecular packing of type II collagen in the fibrils of the lamprey notochord sheath. This is the first finding of a crystalline structure in any collagen other than type I.The lamprey notochord sheath has a composition similar to that of cartilage, with type II collagen, a minor collagen component with 1α, 2α and 3α chains, and cartilage-like proteoglycan. The high degree of orientation of fibrils in the notochord makes it possible to use X-ray diffraction to determine collagen fibril organization in this type II-containing tissue. The low angle equatorial scattering shows the fibrils are all about 17 nm in diameter and have an average center-to-center separation of 31 nm. These results are supported by electron microscope observations. A set of broad equatorial diffraction maxima at higher angles represents the sampling of the collagen molecular transform by a limited crystalline lattice, extending over a lateral dimension close to the diameter of one fibril. This indicates that each 17 nm fibril contains a crystalline array of molecules and, although a unit cell is difficult to determine because of the broad overlapping reflections, it is clear that the quasi-hexagonal triclinic unit cell of type I collagen in rat tail tendon is not consistent with the data. The meridional diffraction pattern showed 26 orders with the characteristic 67 nm periodicity found for tendon. However, the intensities of these reflections differ markedly from those found for tendon and cannot be explained by an unmodified gap/ overlap model within each 67 nm period. Both X-ray diffraction and electron microscope data indicate a low degree of contrast along the fibril axis and are consistent with a periodic binding of a non-collagenous component in such a way as to obscure the gap region.     

THE CRYSTALLINE CELL WALL OF TETRASELMIS CONVOLUTAE (CHLOROPHYTA): A FREEZE FRACTURE ANALYSIS1

A freeze fracture analysis of the cell wall of Tetraselmis convolutae (Parke et Manton) revealed the existence of a crystalline median layer consisting of regular repeating subunits of 27 nm. These circular subunits lie in curved, interlocking, longitudinal rows with some irregular discontinuities appearing in the subunit pattern of the crystalline lattice. A comparison with the cell walls of other green algal flagellates is presented, revealing similarities and suggesting evolutionary patterns.     

High resolution scanning electron microscopy of plasmodesmata

Symplastic transport occurs between neighbouring plant cells through functionally and structurally dynamic channels called plasmodesmata (PD). Relatively little is known about the composition of PD or the mechanisms that facilitate molecular transport into neighbouring cells. While transmission electron microscopy (TEM) provides 2-dimensional information about the structural components of PD, 3-dimensional information is difficult to extract from ultrathin sections. This study has exploited high-resolution scanning electron microscopy (HRSEM) to reveal the 3-dimensional morphology of PD in the cell walls of algae, ferns and higher plants. Varied patterns of PD were observed in the walls, ranging from uniformly distributed individual PD to discrete clusters. Occasionally the thick walls of the giant alga Chara were fractured, revealing the surface morphology of PD within. External structures such as spokes, spirals and mesh were observed surrounding the PD. Enzymatic digestions of cell wall components indicate that cellulose or pectin either compose or stabilise the extracellular spokes. Occasionally, the PD were fractured open and desmotubule-like structures and other particles were observed in their central regions. Our observations add weight to the argument that Chara PD contain desmotubules and are morphologically similar to higher plant PD.     

SECRETION OF PHENOLIC SUBSTANCES INTO THE ZYGOTE WALL AND CELL PLATE IN EMBRYOS OF HORMOSIRA AND ACROCARPIA (FUCALES, PHAEOPHYCEAE)

We have used light and electron microscopy to study the secretion of phenolic substances into the primary walls and cross walls of zygotes and embryos of the brown algae Hormosira banksii (Turner) Decaisne (Hormosiraceae) and Acrocarpia paniculata (Turner) Areschoug (Cystoseiraceae). Phenolic compounds packaged within physodes are a major cytoplasmic component of brown algae. Physodes accumulate at the zygote periphery early in development and are secreted into the primary zygote wall. At germination, physodes accumulate at the rhizoid tip. The first sign of impending cytokinesis is the lining up of these phenolic vesicles in the plane of cell division. This precedes the arrival of other wall constituents. Physodes, together with other wall components, contribute to the development of the cell plate. We conclude that phenolic compounds play an important role in cell-wall construction in these species.     

Crystalline tubes of myosin subfragment-2 showing the coiled-coil and molecular interaction geometry

We have produced crystalline tubes of chicken breast myosin long subfragment-2 that show order to resolutions better than 2 nm. The tubes were formed from a thin sheet in which the myosin long subfragment-2 molecules were arranged on an approximately rectangular crystalline lattice with a = 14.1 +/- 0.2 nm and b = 3.9 +/- 0.1 nm in projection. Shadowing indicated that the tube wall was approximately 7 nm thick and that the sheets from which it was formed followed a right-handed helix. Superposition of the lattices from the top and bottom of the tube produced a moire pattern in negatively stained material, but images of single sheets were easily obtained by computer image processing. Although several molecules were superimposed perpendicular to the plane of the sheet, the modulation in density due to the coiled-coil envelope was clear, indicating that the coiled-coils in these molecules were in register (or staggered by an even number of quarter pitches). In projection the coiled-coil had an apparent pitch of 14.1 nm (the axial repeat of the unit cell), but the small number of molecules (probably four) superimposed perpendicular to the plane of the sheet meant that pitches within approximately 1 nm of this value could have shown a modulation. Therefore, a more precise determination of the coiled-coil pitch must await determination of the sheet's three-dimensional structure. The coiled-coils of adjacent molecules within the plane of the sheet were staggered by an odd number of quarter pitches. This arrangement was similar to that between paramyosin molecules in molluscan thick filaments and may have features in common with other coiled-coil protein assemblies, such as intermediate filaments. Each molecule in the crystal had two types of neighbor: one staggered by an odd number of quarter pitches and the other by an even number of quarter pitches, as has been proposed for the general packing of coiled-coils (Longley, W., 1975, J. Mol. Biol., 93:111-115). We propose a model for the detailed packing within the sheet whereby molecules are inclined slightly to the plane of the sheet so that its thickness is determined by the molecular length.     

Plant and algal cell walls: diversity and functionality

Background

Although plants and many algae (e.g. the Phaeophyceae, brown, and Rhodophyceae, red) are only very distantly related they are united in their possession of carbohydrate-rich cell walls, which are of integral importance being involved in many physiological processes. Furthermore, wall components have applications within food, fuel, pharmaceuticals, fibres (e.g. for textiles and paper) and building materials and have long been an active topic of research. As shown in the 27 papers in this Special Issue, as the major deposit of photosynthetically fixed carbon, and therefore energy investment, cell walls are of undisputed importance to the organisms that possess them, the photosynthetic eukaryotes (plants and algae). The complexities of cell wall components along with their interactions with the biotic and abiotic environment are becoming increasingly revealed.

Scope

The importance of plant and algal cell walls and their individual components to the function and survival of the organism, and for a number of industrial applications, are illustrated by the breadth of topics covered in this issue, which includes papers concentrating on various plants and algae, developmental stages, organs, cell wall components, and techniques. Although we acknowledge that there are many alternative ways in which the papers could be categorized (and many would fit within several topics), we have organized them as follows: (1) cell wall biosynthesis and remodelling, (2) cell wall diversity, and (3) application of new technologies to cell walls. Finally, we will consider future directions within plant cell wall research. Expansion of the industrial uses of cell walls and potentially novel uses of cell wall components are both avenues likely to direct future research activities. Fundamentally, it is the continued progression from characterization (structure, metabolism, properties and localization) of individual cell wall components through to defining their roles in almost every aspect of plant and algal physiology that will present many of the major challenges in future cell wall research.     

Three-dimensional architecture of the cell sheath and septa of Methanospirillum hungatei.

The methanogenic bacterium Methanospirillum hungatei exists as filaments which have a very unusual cell wall architecture, comprising a long cylindrical sheath within which there may be many individual cells arranged in a line. The sheath has a two-dimensional crystalline structure, and the cells are separated within the tube by septa which also have a crystalline structure. We have used computer image processing of tilted-view electron micrographs to analyze the structure in negative stains of both of these components in three dimensions. The repeating unit of the sheath consists of four approximately spherical domains ca. 2.5 nm in diameter arranged in a row. Based on observations of the type of lattice imperfections that occur, we suggest that each of the domains represents a separate polypeptide subunit and that the subunits are incorporated into the wall one by one. The septa are circular plates of remarkably constant size. They are normally found as double layers. They are hexagonally symmetrical and consist of trimerically associated subunits which interact about dimer axes to form an open network containing large pores ca. 15 nm in diameter.     

Ultrastructure and ontogeny of a new type of eyespot in dinoflagellates

Summary Ultrastructure and ontogeny of a new type of eyespot in dinoflagellates is described. A marine tidal poolGymnodinium natalense is found to possess a highly organized eyespot whose structure is unique among dinoflagellates. The eyespot is rectangular in ventral view, C-shaped in apical view, and is located posterior to the sulcus. The eyespot is independent of the chloroplast and consists of several (typically six) layers of hemi-cylindrical walls which are concentrically arranged with narrow spacing between them. Each hemicylindrical wall is enclosed by a single unit membrane and is composed of many regularly arranged rectangular crystalline bricks. These crystalline bricks are produced in small vesicles which are formed in the invaginations of the chloroplast. The vesicles containing newly formed crystalline bricks are then transported to the sulcal area to assemble the eyespot. The crystalline bricks are arranged in a neat row within the vesicle termed “eyespot forming vesicle” (EFV), which is located near the sulcus. The hemi-cylindrical wall is constructed within the EFV. Based on the structure of the eyespot, viz. consisting of concentric multi-layered walls, the eyespot is thought to act as a quarter-wave stack antenna.     

Preliminary X-ray crystallographic analysis of canine parvovirus crystals

The first diffraction pattern of a crystalline single-stranded DNA virus has been obtained. Canine parvovirus was crystallized in a monoclinic P21 unit cell with a = 264.4 A, b = 350.3 A, c = 267.8 A and beta = 90.86 degrees (1 A = 0.1 nm). The diffraction pattern extends to at least 2.8 A resolution. Packing of the particles suggests that they have a diameter around 257 A, in excellent agreement with the reported molecular weight of 5.5 x 10(6).     

Out-of-plane orientation of cellulose elementary fibrils on spruce tracheid wall based on imaging with high-resolution transmission electron microscopy

Main conclusion

A 3D model of the tracheid wall has been proposed based on high-resolution cryo-TEM where, in contrast to the current understanding, the cellulose elementary fibrils protrude from the cell wall plane. The ultrastructure of the tracheid walls of Picea abies was examined through imaging of ultrathin radial, tangential and transverse sections of wood by transmission electron microscopy and with digital image processing. It was found that the elementary fibrils (EFs) of cellulose were rarely deposited in the plane of the concentric cell wall layers, in contrast to the current understanding. In addition to the adopted concept of longitudinal fibril angle, EFs protruded from the cell wall plane in varying angles depending on the layer. Moreover, the out-of-plane fibril angle varied between radial and tangential walls. In the tangential S₂ layers, EFs were always out-of-plane whereas planar orientation was typical for the S₂ layer in radial walls. The pattern of protruding EFs was evident in almost all axial and transverse images of the S₁ layer. Similar out-of-plane orientation was found in the transverse sections of the S₃ layer. A new model of the tracheid wall with EF orientation is presented as a summary of this study. The outcome of this study will enhance our understanding of the elementary fibril orientation in the tracheid wall.     

On the mechanism of uranium binding to cell wall of <Emphasis Type="Italic">Chara fragilis</Emphasis>

Biosorption of uranium from nuclear waste liquids and contaminated surface waters and soils has recently attracted special interest. However, the detailed mechanism of uranium uptake by plants is not well understood. The aim of this work is to investigate the role of cell wall components of the freshwater alga Chara fragilis in uranium sequestration from its solution. Three types of algae preparations: extract of cell wall polysaccharides, dried and live algae were subjected to uranium solutions of different concentration and pH. FTIR and X-ray diffraction were used to assess both potential binding sites and the form of the uranyl sequestered by algae. Sorption of uranium by live and dry algae shows remarkable differences both in terms of overall uptake and mechanisms involved. All experiments are consistent with the conclusion that coprecipitation of uranyl species with CaCO3 is the major binding mechanism in uranium sequestration by Chara fragilis, while the direct exchange of Ca2+ with UO22+ has a minor role. Live algae are twice as efficient in sequestering uranium from solution than dried ones due to the formation of different crystalline forms such as aragonite and rutherfordine forming in live algae in the presence of the uranyl species in solution. It therefore appears that metabolic processes such as photosynthesis, most likely through the regulation of pH, play a key role in the uranium uptake by plants. Further understanding of the complex mechanism of metabolic control of the uranium uptake by plants is needed before the planning of bioremediation of this element.     

Nitrogen in cell walls of sclerophyllous leaves accounts for little of the variation in photosynthetic nitrogen-use efficiency

Photosynthetic rate per unit nitrogen generally declines as leaf mass per unit area (LMA) increases. To determine how much of this decline was associated with allocating a greater proportion of leaf nitrogen into cell wall material, we compared two groups of plants. The first group consisted of two species from each of eight genera, all of which were perennial evergreens growing in the Australian National Botanic Gardens (ANBG). The second group consisted of seven Eucalyptus species growing in a greenhouse. The percentage of leaf biomass in cell walls was independent of variation in LMA within any genus, but varied from 25 to 65% between genera. The nitrogen concentration of cell wall material was 0.4 times leaf nitrogen concentration for all species apart from Eucalyptus , which was 0.6 times leaf nitrogen concentration. Between 10 and 30% of leaf nitrogen was recovered in the cell wall fraction, but this was independent of LMA. No trade-off was observed between nitrogen associated with cell walls and the nitrogen allocated to ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco). Variation in photosynthetic rate per unit nitrogen could not be explained by variation in cell wall nitrogen.     

A Lichen Lectin Specifically Binds to the α-1,4-Polygalactoside Moiety of Urease Located in the Cell Wall of Homologous Algae

A lectin from the lichen Evernia prunastri developing arginase activity (EC. 3.5.3.1) binds to the homologous algae that contain polygalactosilated urease (EC. 3.5.1.5) in their cell walls acting as a lectin ligand. The enzyme bound to its ligand shows to be inactive to hydrolyze of arginine. Hydrolysis of the galactoside moiety of urease in intact algae with α-1,4-galactosidase (EC. 3.2.1.22) releases high amount of D-galactose and impedes the binding of the lectin to the algal cell wall. However, the use of β-,4-galactosidase (EC.3.2.1.23) releases low amounts of D-galactose from the algal cell wall and does not change the pattern of binding of the lectin to its ligand. The production of glycosilated urease is restricted to the season in which algal cells divide and this assures the recognition of new phycobiont produced after cell division by its fungal partner.Key Words: arginase, cell wall, evernia prunastri, lectin ligand, phycobiont, urease     

Mobility-resolved 13C-NMR spectroscopy of primary plant cell walls

Primary plant cell walls contain highly hydrated biopolymer networks, whose major chemistry is known but whose relationship to architectural and mechanical properties is poorly understood. Nuclear magnetic resonance spectroscopy has been used to characterize segmental mobilities via relaxation and anisotropy effects in order to add a dynamic element to emerging models for cell wall architecture. For hydrated onion cell wall material, single pulse excitation revealed galactan (pectin side chains), provided that dipolar decoupling was used, and some of the pectin backbone in the additional presence of magic angle spinning. Cross-polarization excitation revealed the remaining pectin backbones, which exhibited greater mobility (contact time dependence, dipolar dephasing) than the cellulose component, whose noncrystalline and crystalline fractions showed no mobility discrimination. ¹HT₂ behavior could be quantitatively interpreted in terms of high resolution observabilities. Mobility-resolved spectroscopy of cell walls from tomato fruit, pea stem, and tobacco leaf showed similar general effects. Nuclear magnetic resonance study of the sequential chemical extraction of onion cell wall material suggests that galactans fill many of the network pores, that extractability of pectins is not dependent on segmental mobility, and that some pectic backbone (and not side chain) is strongly associated with cellulose. Analysis of the state of cellulose in four hydrated cell walls suggests a noncrystalline content of 60–80% and comparable amounts of Iα and Iβ polymorphs in the crystalline fraction. Comparison with micrographs for onion cell walls shows that noncrystalline cellulose does not equate to chains on fibril surfaces, and chemical shifts show that fully solvated cellulose is not a significant component in cell walls. © 1996 John Wiley & Sons, Inc.