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.     

THE CELL WALL OF SCENEDESMUS QUADRICAUDA

Bisalputra, T., and T. E. Weier. (U. California, Davis.) The cell wall of Scenedesmus quadricauda. Amer. Jour. Bot. 50(10): 1011–1019. Illus. 1963.—Fine structure of the cell wall of Scenedesmus quadricauda fixed in both KMnO₄ and osmium tetroxide is described. The cell wall consists of 3 layers: the inner cellulosic layer which delimits individual cells; the outer pectic layer which binds the cells of the coenobium together; and a thin middle layer, bounded by membranes on either side, which is electron-dense in osmium-fixed material but of medium electron density in KMnO₄. The structure of the outer pectic layer is similar in both fixatives; it consists of a hexagonal network of electron-dense material on the surface, and a system of tubules or “props” which radiate out from the middle layer of the wall to support the net. The pectic layer appears in the daughter coenobia before their liberation from the parent colony.     

SUGAR COMPOSITION OF THE CELL WALL AND THE TAXONOMY OF CHLORELLA (CHLOROPHYCEAE)1

Cell walls of forty Chlorella strains covering all species of the Algal Collection of Göttingen (C. fusca var. vacuolata, C. kessleri, C. luteoviridis, C. minutissima, C. protothecoides, C. saccharophila, C. sorokiniana, C. vulgaris, and C. zofingiensis) were compared. The nine species were divided into two groups according to the major sugar in the rigid wall. The first group had a glucose-mannose-rigid wall and included C. fusca var. vacuolata, C. luteoviridis, C. minutissima, C. protothecoides, C. saccharophila, and C. zofingiensis. The second group, with a glucosamine-rigid wall, included C. kessleri, C. sorokiniana, and C. vulgaris. Chlorella strains of the nine species were further classified by constituent sugars, ruthenium red stainability, and anisotropy of the cell walls.     

THE CHEMICAL COMPOSITION OF THE CELL WALL OF CHLAMYDOMONAS GYMNOGAMA AND THE CONCEPT OF A PLANT CELL WALL PROTEIN

Cell walls of Chlamydomonas gymnogama, shed during sexual mating, were collected and analyzed. Ultrastructural examination indicates that the walls are free of cytoplasmic contamination and that they exhibit a regular lamellate structure. The walls are composed of glycoprotein rich in hydroxyproline. The hydroxyproline is linked glycosidically to a mixture of heterooligosaccharides composed of arabinose and galactose. Altogether, the glycoprotein complex accounts for at least 32% of the wall. The amino acid composition of the walls is extraordinarily similar in widely different plant species. The implications of these similarities as well as the widespread occurrence of these glycoproteins are discussed.     

DNA POLYMORPHISM WITHIN THE WH7803 SEROGROUP OF MARINE SYNECHOCOCCUS SPP. (CYANOBACTERIA)1,2

Genetic differences among ten strains of chroococcoid cyanobacteria (Synechococcus spp.) were identified by Southern blot hybridization. Data on shared number of restriction fragment length polymorphisms were used to identify the pattern and degree of genetic relatedness among the strains by two different methods of phylogenetic analysis. All the marine strains in the study contained phycoerythrin (PE) and cross-reacted with antisera directed against strain WH7803. Five contained a PE composed of phycourobilin (PUB) and phycoerythrobilin (PEB) Chromophores, and three contained a PE composed of only PEB chromophores. Two freshwater strains which do not contain PE and do not cross-react with the anti-WH7803 serum were included in the study for comparison. Dollo Parsimony analysis and cluster analysis showed that the WH7803 serogroup includes at least four widely separated genetic lineages. Strains within each lineages were closely related but the differences between lineages were as great as those between any of the marine lineages and the freshwater lineage. Strains cultured simultaneously from the same water mass were associated with different lineages. Thus, we conclude that natural assemblages of marine. Synechococcus are, at least occasionally, composed of individuals as genetically distinct from each other as members of different species or genera in other taxa.     

THE COMPOSITION AND PHYLOGENETIC SIGNIFICANCE OF THE MOUGEOTIA (CHAROPHYCEAE) CELL WALL1

The two-layered, fibrillar cell wall of Mougeotia C. Agardh sp. consisted of 63.6% non-cellulosic carbohydrates and 13.4% cellulose. The orientation of cellulose microfibrils in the native cell wall agrees with the multinet growth hypothesis, which has been employed to explain the shift in microfibril orientation from transverse (inner wall) toward axial (outer wall). Monosaccharide analysis of isolated cell walls revealed the presence of ten sugars with glucose, xylose and galactose most abundant. Methylation analysis of the acid-modified, 1 N NaOH insoluble residue fraction showed that it was composed almost exclusively of 4-linked glucose, confirming the presence of cellulose. The major hemicellulosic carbohydrate was semi-purified by DEAE Sephacel (Cl^?) anion-exchange chromatography of the hot 1 N NaOH soluble fraction. This hemicellulose was a xylan consisting of a 4-xylosyl backbone and 2,4-xylosyl branch points. The major hot water soluble neutral polysaccharide was identified as a 3-linked galactan. Mougeotia cell wall composition is similar to that of (Charophyceae) and has homologies with vascular plant cell walls. Our observations support transtructural evidence which suggests that members of the Charophyceae represent the phylogenetic line that gave rise to vascular plants. Therefore, the primary cell walls of vascular plants many have evolved directly from structures typical of the filamentous green algal cell walls found in the Charophyceae.     

DIFFERENTIATION,ORGANOGENESIS, AND THE TECTONICS OF CELL WALL ORIENTATION. III. THEORETICAL CONSIDERATIONS OF CELL WALL MECHANICS

This paper is concerned with the relationship between cell wall orientation in dividing tissues and stress. An examination of the possible orientations of a cell-plate in a cell under axial stress reveals that only one orientation passing through the center of the cell can be completely free from shear stress, thus providing a favored plane for the positioning of an hypothetical, shear-sensitive, cell-plate precursor. From this principle of shear-free partitioning, and the three basic rules of stress behavior along free boundaries, it is possible to predict certain features of cell wall orientation along free edges and epidermal surfaces and in some simple apices. The possible significance of the widespread phenomenon of axillary induction of bud formation is discussed in terms of the generation of stresses at the base of the axillant leaf, and the general efficacy of mechanical stress as a spontaneously arising morphogenetic trigger is considered.     

AN INVESTIGATION OF THE CELL WALL COMPONENTS OF ACTINOCYCLUS SUBTILIS (BACILLARIOPHYCEAE)1

The frustule of Actinocyclus subtilis (Greg.) Ralfs has been examined in detail with light and electron microscopy. The complex valve structure can only he appreciated fully by means of thin-sections. The loculate areola is closed to the inside by a basket of organic material, which is supported by a system of radiating, lightly silicified ribs. The basket is easily destroyed by acid cleaning, thus leaving an open hole (foramen). The loculus is connected to the outside by a system of interconnecting channels, whose external openings are too small to be resolved by the SEM. Between the locules of the valve face is a complex system of bullulae. The labiate processes are slightly tilted from the vertical axis vis à vis the valve mantle. Fibrillar material and mucilage are present within each labiate process, observations not reported previously. Histochemistry indicates that acid mucopolysaccharides are present within the lumen of the labiate process. We propose that the secretion of mucilage is related to the rotational, but directed movement noted in this centric diatom. The pseudonodulus is a solid silica plate subtended on the inside by a disc of organic material external to the cytoplasm. The relationship between Actinocyclus, Hemidiscus, Roperia, Charcotia and Azpeitia is discussed based on these new findings of valve morphology. Also the relationship between the families Hemidiscaceae and Coscinodiscaceae is discussed.     

CELL WALL CHEMISTRY AND FINE STRUCTURE IN LEPTOIDS OF DENDROLIGOTRICHUM (BRYOPHYTA): THE END WALL

Leptoids (sieve elements) of Dendroligotrichum are characterized by a highly oblique end wall which is composed of cellulose (birefringent; IKI-H₂SO₄-positive), polyuronides (toluidine blue-positive), pectins (hydroxylamine-positive) and natural aldehydes (silver hexamine and silver proteinate-positive). Cytochemically the end wall appears identical to the unevenly thickened lateral wall. Electron cytochemical localization of aldehydes with silver proteinate reveals two distinct wall layers in comparison to the 3-layered lateral wall. Plasmodesmata are present in the end wall with a frequency of 15-20 per μm². A characteristic feature of end wall plasmodesmata is an expanded median cavity which is 0.12-0.15 μm in diameter. Frequently an electron-dense substance, whose chemical nature and origin are unknown, occludes the plasmodesmata.     

THE CELL WALL OF COSMARIUM BOTRYTIS12

The cell wall of Cosmarium botrytis was studied through the use of the freeze-etch technique. The cell wall consists of many thin layers. Fracturing along one layer reveals the positioning of the wall sculpturing, wall pores, and wall microfibrils. The individual microfibrils are grouped together in bands of parallel oriented fibrils. The different bands of parallel microfibrils were apparently arranged at random angles with regard to each other. Small particles may also be present in the cell walls. The cell wall pore unit of Cosmarium botrytis was studied through the use of scanning, freeze-etching, and thin sectioning techniques. The pore sheaths, on the outside of the cell wall, form a collar around the mouth of each pore. The pore sheath is composed of needle-like fibrils radiating outward from the pore. A pore channel traverses the cell wall and leads to a complex pore bulb region between the cell wall and the plasmalemma. The pore bulb contains many small fibrils which radiate toward the plasmalemma from a number of net-like fibril layers which in turn merge into a very electron dense region near the base of the pore.     

THE PECTIC LAYER OF THE CELL WALL OF SCENEDESMUS QUADRICAUDA

Further evidence from shadow casting techniques of the empty cells of Scenedesmus quadricauda confirms the structure of the pectic layer presented in an earlier work which was based entirely on reconstruction from thin sections. The structure of the net and props and their relationship are clearly seen and the inner boundary of the pectic layer is demonstrated by staining techniques.     

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.     

COMPOSITION AND SYNTHESIS OF THE PECTIN AND PROTEIN COMPONENTS OF THE CELL WALL OF CLOSTERIUM ACEROSUM (CHLOROPHYTA)

Closterium acerosum Ehrenberg (Chlorophyta) possesses a trilayered cell wall consisting of an outer tri-laminate stratum, a fibrous middle layer, and a thick inner fibrous layer. The outermost layer has a series of external parallel ridges and valleys. At the bases of the valleys are the wall pores, the site of mucilage release. Pure fractions of cell walls were isolated and inclusive pectin and wall protein fractions were extracted and characterized. Two pectin-like fractions were isolated: a CDTA-extracted polymer consisting of 60.1% galacturonic acid and a Na₂CO₃-extracted fraction consisting of 39.9% galacturonic acid. Two major protein fractions, one with a molecular mass of 23.5 kDa and one with a molecular mass of 28.5 kDa, were isolated by preparative gel electrophoresis. The former was glycine-rich, whereas the latter contained both significant amounts of glycine and hydroxyproline. Antibodies were raised to both the pectin fractions and the 23.5-kDa wall protein fraction. Immunocytochemical labeling of whole cells and wall fragments using antibodies raised against CDTA and Na₂CO₃ extracts showed that these pectin-like components were found throughout the wall strata and were more concentrated at the polar tips, the site of new wall synthesis in growing semicells. Immunogold labeling showed that their production was focused on the trans- Golgi network of the Golgi apparatus. Immunolabeling with an antibody raised against the 23.5-kDa glycine-rich wall protein showed close association of the protein with the wall pores. Similarly, immunogold labeling revealed that the protein was processed throughout the entire Golgi body even when large mucilage-containing vesicles were being processed. The roles of the secretory apparatus and putative spitzenkorper-like regions of the cell are discussed.     

WATER PERMEABILITY OF THE CELL WALL IN NITELLA

1. A method has been developed to measure the hydraulic conductivityof the wall of the internodal cell of Nitella flexilis.
2. Therate of water penetration through the cell wall varies linearlywith the hydrostatic pressure difference between the two sidesof the wall, showing that water permeability of the cell wallremains independent of the pressure difference applied.
3. Waterpermeability of the cell wall is inversely proportionalto itsthickness It is 30µµmin^–3{dot}atm^–3when the thickness of the wall is 10 µ.
4. Water permeabilityof the cell wall is the same for inward andoutward water flow.The polar water permeability of the entiremembrane system (walland protoplasmic part) of the living celldemonstrated by KAMIYAand TAZAWA (1) is, therefore, due tothe living protoplasmicpart.
5. The ratio of the inward to outward permeability constantsofthe protoplasmic layer alone is higher than that of the entiremembrane system composed of protoplasmic layer and cell wall.

¹ Dedicated to Prof. H. TAMIYA on the occasion of his 60th birthday.The present work was supported in part by a Grant-in-Aid forFundamental Scientific Research from the Ministry of Education. ² Present address: Sh?in Women's College, Kobe. (Received July 21, 1962; )     

THE STRUCTURE OF THE PRIMARY EPIDERMAL CELL WALL OF AVENA COLEOPTILES

The primary walls of epidermal cells in Avena coleoptiles ranging in length from 2 to 40 mm. have been studied in the electron and polarizing microscopes and by the low-angle scattering of x-rays. The outer walls of these cells are composed of multiple layers of cellulose microfibrils oriented longitudinally; initially the number of layers is between 10 and 15 but this increases to about 25 in older tissue. Where epidermal cells touch, these multiple layers fuse gradually into a primary wall of the normal type between cells. In these radial walls, the microfibrils are oriented transversely. Possible mechanisms for the growth of the multilayered outer wall during cell elongation are discussed.     

THE CELL WALL (THECA) OF TETRASELMIS STRIATA (CHLOROPHYTA): MACROMOLECULAR COMPOSITION AND STRUCTURAL ELEMENTS OF THE COMPLEX POLYSACCHARIDES

Isolated cell walls (thecae) from the scaly flagellate green alga Tetraselmis striata Butcher contain the unusual 2-keto-sugar acids 3-deoxy-manno-2-octulosonic acid (Kdo), 3-deoxy-5-O-methyl-manno-2-octulosonic acid (5OMeKdo), and 3-deoxy-lyxo-2-heptulosaric acid (Dha). In addition, galacturonic acid, galactose, gulose, and arabinose are present. EDTA-extraction yielded an insoluble fraction that retains the shape of the cell walls and contains no 2-keto-sugar acids. Methylation analysis demonstrated the presence of terminal hexose, GalA, Dha, and Kdo as well as 2-substituted hexose, 4-or 8-substituted Kdo, and 4,8-disubstituted Kdo. However, most of the carbohydrate material (about 60%) was not methylated. Periodate oxidation of the cell wall preparation showed the presence of 2-substituted Gul, 4- or/and 5-substituted and 7- or/and 8-substituted Kdo, which is in agreement with the methylation analysis. Again, a significant amount of carbohydrate material was not degraded, indicating complex substitution patterns. Oligosaccharides were generated by partial hydrolysis and fractionated using gel permeation chromatography and high-pH anion-exchange chromatography. Oligosaccharides contained either GalA and Kdo, or Gal, Kdo, Dha, and Gul, respectively. The structure of a GalA and Kdo containing disaccharide was established using ¹ H NMR spectroscopy.