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.     

STUDIES ON THE ALGAL CUTICLE1

A chemically resistant cuticle fraction was isolated from 5 phaeophycean, 1 rhodophycean, and 11 chlorophycean marine algae using acid treatment alone, or acid treatment followed by leaching in cupra-ammonium. In Cladophora rupestris and Chaetomorpha melagonium this fraction consists of several alternate microfibrillar and amorphous layers similar in appearance to those seen in innermost carbohydrate-rich regions and amount to about 1/10 or more of the cell wall thickness. In Porphyra umbilicalis and Padina vickersiae it is a single layer less than I μ thick, accounting for 1/50–1/100 of the cell wall in Porphyra, and 1/5–1/10 of the cell wall in Padina. The cuticle fractions of all 4 algae contain surprisingly large amounts of protein (about 70% in Cladophora and 80% in Porphyra). Similarities in the behavior of cuticles obtained from the other 12 species studied suggest that they may have a similar protein-rich composition.     

Hydroxyproline and Peroxidases in Cell Walls of Pisum sativum: Regulation by Ethylene

Purified cell-wall preparations from the epicotyl of etiolatedPisum sativum contain covalently bound peroxidases and hydroxyproline-richproteins. Towards the end of cell elongation there is a largerise in these wall components and thereafter a continuing slowrise which is associated with increasing age of tissue. Ethyleneat concentrations of 0.1 ppm or more increases both peroxidaseactivity and hydroxyproline levels in the walls, the greatestresponse occurring in immature tissue including the apical hook.Growth of these tissues is highly sensitive to ethylene whichcauses an inhibition of elongation in extending cells and anenhanced lateral cell expansion. We suggest that the effectsof ethylene on wall-bound peroxidase and hydroxyproline areimplicated in the ethylene regulation of cell growth. The covalently bound wall peroxidase was found to be extremelystable and to contain unique isoenzymes which do not occur ineither the cytoplasm or in the peroxidase which is ionicallybound to walls. Ethylene increases peroxidase activity in boththe cytoplasmic and the ionically bound wall fractions, butthere is little or no increase in their hydroxyproline content.The possible relationships between covalently bound wall peroxidaseand hydroxyproline are discussed and we speculate that thisperoxidase may be involved in the hydroxylation of proline inthe walls.     

The comparative aspects of cell wall chemistry in the green algae (chlorophyta)

Summary The origin of a cell wall was an event of fundamental importance in the evolution of plants. In the green algae, cell walls apparently had independent origins in at least three lines of evolution. In this paper, the components of the cell wall were determined and compared in four filamentous green algae representing the charophycean, chlorophycean and ulvacean evolutionary lines. The walls of all four have hydroxyproline-containing proteins which separate into five or six bands upon SDS gel electrophoresis. Variation does exist, with the charophyte possessing fast moving electrophoretic bands and high hydroxyproline content, the chlorophytes having intermediate movement of bands and lower hydroxyproline content, and the ulvacean representative possessing slow moving bands and a very low, if not questionable, hydroxyproline and saccharide content. Qualitative and quantitative estimates of wall proteins and sugars have been determined and compared. A hypothetical scheme of cell wall evolution based on these data, those of previous analyses, and recent phylogenetic schemes is presented. Although sound conclusions cannot be made until more information is available, the scheme might help to emphasize the areas most in need of additional research.This work was supported by National Science Foundation Grant DEB 78-03554.     

Hydroxyproline-rich cell wall protein (extensin): Biosynthesis and accumulation in growing pea epicotyls

Plant cell walls contain a glycoprotein component rich in the otherwise rare amino acid hydroxyproline. We examined the synthesis and accumulation of wall hydroxyproline during different states of elongation growth in pea epicotyls. Light-grown peas contained more wall hydroxyproline than their taller, dark-grown counterparts. When elongation was studied by marking growing stems in situ, there was a marked accumulation of wall hydroxyproline coincident with the cessation of elongation. Dividing and elongating regions of the epicotyl showed less wall hydroxyproline than did regions where elongation was no longer occurring.Hydroxyproline biosynthesis was examined by incubation of excised sections of tissues in various growth states in ¹⁴C-proline. The extent of conversion of these residues to ¹⁴C-hydroxyproline served as a measure of the rate of hydroxyproline synthesis. This rate was highest in tissues which had ceased elongation. The low rate of hydroxyproline synthesis in dividing and elongating cells was probably not due to the inability to hydroxylate peptidyl proline or to secrete proteins.These data show a positive correlation between the synthesis and accumulation of cell wall hydroxyproline and the cessation of cell elongation in pea epicotyls.     

Chemical and in situ characterization of macromolecular components of the cell walls from the green seaweed Codium fragile

A comprehensive analysis of the carbohydrate-containing macromolecules from the coencocytic green seaweed Codium fragile and their arrangement in the cell wall was carried out. Cell walls in this seaweed are highly complex structures composed of 31% (w/w) of linear (1-->4)-beta-D-mannans, 9% (w/w) of pyruvylated arabinogalactan sulfates (pAGS), and low amounts of hydroxyproline rich-glycoprotein epitopes (HRGP). In situ chemical imaging by synchrotron radiation Fourier transform infrared (SR-FTIR) microspectroscopy and by immunolabeling using antibodies against specific cell wall carbohydrate epitopes revealed that beta-d-mannans and pAGS are placed in the middle part of the cell wall, whereas HRGP epitopes (arabinogalactan proteins (AGPs) and extensins) are located on the wall boundaries, especially in the utricle apical zone. pAGS are sulfated at C-2 and/or C-4 of the 3-linked beta-L-arabinopyranose units and at C-4 and/or C-6 of the 3-linked beta-D-galactopyranose residues. In addition, high levels of ketals of pyruvic acid were found mainly at 3,4- of some terminal beta-D-Galp units forming a five-membered ring. Ramification was found at some C-6 of the 3-linked beta-D-Galp units. In agreement with the immunolabeled AGP epitopes, a nonsulfated branched furanosidic arabinan with 5-linked alpha-L-Araf, 3,5-linked alpha-L-Araf, and terminal alpha-L-Araf units and a nonsulfated galactan structure composed of 3-(3,6)-linked beta-D-Galp residues, both typical of type-II AG glycans were found, suggesting that AGP structures are present at low levels in the cell walls of this seaweed. Based on this study, it is starting to emerge that Codium has developed unique cell wall architecture, when compared, not only with that of vascular plants, but also with other related green seaweeds and algae.     

Synthesis and Transport of Hydroxyproline-rich Components in Suspension Cultures of Sycamore-Maple Cells

Plant cell walls contain a glycoprotein rich in hydroxyproline. To determine how Acer pseudoplatanus L. cells transport this glycoprotein to the wall, the pulse-chase technique was used to follow changes in specific radio-activity of hydroxyproline and proline in isolated, mitochondrial, Golgi, microsomal, soluble protein, and wall fractions. The turnover rates or changes in specific radioactivity of cytoplasmic hydroxyproline in these cell fractions indicated that the bulk of this hydroxyproline was transferred not by the Golgi apparatus but by a smooth membranous component.     

CELL‐WALL POLYMER MAPPING IN THE COENOCYTIC MACROALGA CODIUM VERMILARA (BRYOPSIDALES,CHLOROPHYTA)1

Cell walls in the coenocytic green seaweed Codium vermilara (Olivi) Chiaje (Bryopsidales, Chlorophyta) are composed of ～32% (w/w) β‐(1→4)‐d‐mannans, ～12% sulfated polysaccharides (SPs), and small amounts of hydroxyproline‐rich glycoprotein‐like (HRGP‐L) compounds of the arabinogalactan proteins (AGPs) and arabinosides (extensins). Similar quantities of mannans and SPs were reported previously in the related seaweed C. fragile (Suringar) Hariot. Overall, both seaweed cell walls comprise ～40%–44% of their dry weights. Within the SP group, a variety of polysaccharide structures from pyruvylated arabinogalactan sulfate and pyruvylated galactan sulfate to pyranosic arabinan sulfate are present in Codium cell walls. In this paper, the in situ distribution of the main cell‐wall polymers in the green seaweed C. vermilara was studied, comparing their arrangements with those observed in cell walls from C. fragile. The utricle cell wall in C. vermilara showed by TEM a sandwich structure of two fibrillar‐like layers of similar width delimiting a middle amorphous‐like zone. By immuno‐ and chemical imaging, the in situ distribution of β‐(1→4)‐d‐mannans and HRGP‐like epitopes was shown to consist of two distinct cell‐wall layers, whereas SPs are distributed in the middle area of the wall. The overall cell‐wall polymer arrangement of the SPs, HRGP‐like epitopes, and mannans in the utricles of C. vermilara is different from the ubiquitous green algae C. fragile, in spite of both being phylogenetically very close. In addition, a preliminary cell‐wall model of the utricle moiety is proposed for both seaweeds, C. fragile and C. vermilara.     

A style-specific 120-kDa glycoprotein enters pollen tubes ofNicotiana alata in vivo

Pistils ofNicotiana alata (Link et Otto) contain an abundant, style-specific glycoprotein (120 kDa) that is rich in hydroxyproline and has both extensin-like and arabinogalactan-protein-like carbohydrate substituents. An antibody specific for the protein backbone of the glycoprotein was used to localise the glycoprotein in both unpollinated and pollinated pistils. The glycoprotein is evenly distributed in the extracellular matrix of the style transmitting tract of unpollinated pistils and, despite the presence of extensin-like carbohydrate substituents, is not associated with the walls of the transmitting tract cells. In pollinated pistils the 120-kDa glycoprotein is concentrated in the extracellular matrix adjacent to pollen tubes, and is also present in the cytoplasm and the cell walls of pollen tubes. Pollen tubes grown in vitro do not contain the 120-kDa glycoprotein unless it is added to the growth medium, suggesting that the 120kDa glycoprotein located in pistil-grown pollen tubes is derived from the extracellular matrix of the transmitting tract.     

THE INCORPORATION OF RADIOACTIVE PROLINE INTO CULTURED CELLS : Interpretations Based On Radioautography and Electron Microscopy

Cultured carrot explants, stimulated to grow rapidly in a medium containing coconut milk, were labeled with radioactive proline. After an initial period of absorption (8 hr for proline-³H; 24 hr for proline-¹⁴C) the tissue was allowed to grow for a further period of 6 days in a similar medium free from the radioactivity. Samples were prepared for electron microscopy and radioautography at the end of the absorption period and also after the further growth. The distribution of the products from the radioactive proline in the cells is shown by high-resolution radioautography and is rendered quantitative for the different regions of the cells. The results show that the combined label, which was present in the form of proline and the hydroxyproline derived from it, was all in the protoplasm, not in the cell walls. Any combined label that appeared to be over the cell walls is shown to be due to scatter from adjacent cytoplasmic sites. Initially the radioactivity was concentrated in nuclei, even more so in nucleoli, but it subsequently appeared throughout the ground cytoplasm and was also concentrated in the plastids. The significance of these observations for the general concept of a plant cell wall protein and for the special problem of growth induction in otherwise quiescent cells is discussed.     

ULTRASTRUCTURE OF SPERMATIUM LIBERATION IN THE MARINE RED ALGA PTILOTA DENSA1

The differentiation of male gametes of the marine red alga Ptilota densa was studied by electron microscopy. Mature primary spermatangia are enveloped by a single cell wall and possess a clearly polar subcellular organization. The nucleus is situated apical to large, striated, fibrous vacuoles which are apparently formed by the repeated fusion of dictyosome vesicles. The transformation and liberation of spermatia from spermatangia involve both the secretion of the fibrous vacuoles at the base of the cell and the subsequent rupturing of the spermatangial cell wall. Liberated spermatia are coated with a thin mucilage layer and contain numerous small vesicles and several mitochondria and dictyosomes. The nucleus is cup-shaped and generally lacks a limiting envelope. These findings are discussed in relation to other light and electron microscopic studies of differentiating spermatangia in red algae.     

Effect of Didymella applanata (Niessl/Sacc.) on proline and hydroxyproline containing cell wall proteins of red raspberry

Structural cell wall proteins and their immobilisation through formation of covalent cross-links belong to important defense response mechanisms. In this work, the D. applanata — induced increase of wall-bound proline and hydroxyproline contents were associated with red raspberry resistance. The effect did not depend on the developmental stage and lignification degree of infected primocanes. Pathogen-induced accumulation of hydroxyproline noted in susceptible plants suggested the post-translational hydroxylation of proline-rich proteins and/or may be of other wall proteins. Moreover, the developmentally related increase of hydroxyproline content was associated with the resistance acquirement noted at the end of the summer period. The increase of cell wall structural protein levels was accompanied by free proline accumulation in the cytosol fraction.     

ENDOSPERM STRUCTURE AND STORAGE RESERVE HISTOCHEMISTRY IN THE PALM,WASHINGTONIA FILIFERA

The endosperm of Washingtonia filifera consists of living cells with the same general cellular structure throughout the seed. The major storage reserves are carbohydrate, stored in the form of thickened walls; lipid, stored as numerous small lipid bodies which fill the cytoplasm; and protein, stored as large, but variably-sized, protein bodies. The protein bodies contain two types of inclusions: prismatically-shaped denser protein crystalloids and small crystalline deposits presumed to be phytic acid. The X-ray microanalysis shows these crystalline inclusions do contain P, Ca, Mg, and Fe. Protein bodies are positively stained with PAS. Nuclei are present in all cells, but stain very palely. Plastids and mitochondria are present, but infrequently seen. The plastids have few, poorly developed membranes. Endoplsasmic reticulum and dictyosomes are lacking. The cell wall is thick except in areas of pit fields and consists of three layers which differ in their staining with toluidine blue and in their ultrastructural characteristics: middle lamella, thickened outer wall, and thin inner wall. All wall layers are positively stained with PAS and calcofluor. Although general structural features of the endosperm in Washingtonia filifera are similar to those in date seeds, the composition of the wall polysaccharides and protein bodies appear to differ somewhat.     

Hydroxyproline in Chiorella and its Metabolism in Tobacco Leaves

Hydroxyproline and other compounds were labeled with C¹⁴ by Chlorella pyrenoidosa supplied with C¹⁴O₂ in the light. The hydroxyproline recovered from a hydrolysate of the algae was administered through the cut bases of tobacco leaves. The leaves formed little proline from the hydroxyproline, but the C¹⁴ label was transferred to a variety of other amino acids. Although hydroxyproline is not abundant in plants, it appears to be an active metabolite.     

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.     

Composition of the cell walls of Nicotiana alata Link et Otto pollen tubes

Cell walls isolated from pollen of Nicotiana alata germinated in vitro contain glucose and arabinose as the predominant monosaccharides. Methylation analysis and cytochemical studies are consistent with the major polysaccharides being a (13)--D-glucan (callose) and an arabinan together with small amounts of cellulose. The cell walls contain 2.8% uronic acids. Alcian blue stains the pollen-tube walls intensely at the tip, indicating that acidic polysaccharides are concentrated in the tip. Synthetic aniline-blue fluorochrome is specific primarily for (13)--D-glucans and stains the pollen-tube walls, except at the tip. Protein (1.5%), containing hydroxyproline (2.4%), is present in the cell wall.     

Chitin in the cell wall of yeasts

Summary According to literature a glucan is the main cell wall constituent of baker's yeast and probably of all yeasts. Chitin is reported in some filamentousEndomycetaceae, but is said to be absent in all other yeasts tested.A modified chitosan sulphate crystallization test for detecting chitin in yeasts and the relevant characteristics of these crystals were described. It was applied to 29 yeast species in addition to baker's and brewer's yeast. ExceptSchizosaccharomyces octosporus all were found to contain chitin as a cell wall constituent. However, the amount was very small, varied in different species and seemed to increase in older cultures.Chitin has also been demonstrated as glucosamine on paper chromatograms of concentrated hydrolysates of the cell wall residue, obtained after treating baker's yeast with boiling dilute alkali and acid.     

CELL WALL CONSTITUENTS OF APHANOTHECE HALOPHYTICA (CYANOPHYTA)1

Some aspects of the cell wall and extracellular polysaccharide (ECPS) of the obligate halophile Aphanothece halophytica Frémy (Chroococcales) have been investigated. Extracellular polysaccharide concentration was found to remain constant on a per cell basis in medium containing from 1–3 M NaCl. The rate of ECPS production remained constant during mid-log growth phase and increased substantially as the culture reached stationary phase. The lipopolysaccharide of this organism was found to possess a low and unusual fatty acid content when compared to other chroococcalean forms. The cell wall appears to contain a typical gram-negative peptidoglycan. The covalently attached protein resembles the envelope protein of extremely halophilic bacteria in its possession of a similar molar percentage of amino acids with lipophilic R-groups and a high acidic amino acid fraction. The ECPS and cell wall fractions of A. halophytica were found to chemically more closely resemble those from other non-halophilic, chroococcalean bluegreen algae than those from the obligately halophilic bacteria.     

Electron-energy-loss spectroscopic imaging of calcium and nitrogen in the cell walls of apple fruits

Changes in texture are an integral part of ripening in most fleshy fruits and these changes are thought to be determined, primarily, by alterations in cell wall structure. Electron energy loss spectroscopy (EELS) imaging was used to obtain quantitative information on the levels of calcium and nitrogen in the cell walls of apple (Malus domestica Borkh. cv. Cox's Orange Pippin) fruits. Samples of fruit cortex were prepared for EELS by high-pressure freezing and molecular distillation drying to minimize loss and redistribution of soluble cell wall components such as calcium. The EELS imaging successfully resolved calcium and nitrogen levels in the middle lamella and primary cell wall. When the elemental compositions of the cell walls of Cox's apples from two sites in the UK were compared at harvest or after 6 months storage, the orchard which always produced consistently firmer fruit had significantly lower levels of cell wall calcium and higher levels of cell wall nitrogen. This result was unexpected since firm texture in apples and other fruits has been commonly associated with elevated levels of fruit calcium. The nitrogen-rich material in the sections used for EELS was insoluble in acidified methanol, indicating that it represented a high-molecular-weight component in the cell wall. Furthermore, total tissue hydroxyproline levels were greatest in material with elevated cell wall nitrogen, suggesting enhanced levels of wall structural proteins in the tissue. These data indicate a correlation between increased amounts of cell wall nitrogen and firm fruit texture. The possible role of cell wall proteins in determining the textural properties of fruit tissue is discussed. Received: 19 November 1998 / Accepted: 28 January 1999