MUCILAGE PROCESSING AND SECRETION IN THE GREEN ALGA CLOSTERIUM. II. ULTRASTRUCTURE AND IMMUNOCYTOCHEMISTRY1

We conducted an ultrastructural and immunocytochemical analysis of the subcellular components involved in mucilage secretion in Closterium. In conventionally fixed cells, the mucilage vesicle appears dense-cored with an electron-dense center surrounded by radiating fibrils. In freeze-substituted cells, the vesicles are highly osmiophilic. These mucilage vesicles are produced from peripheral swellings of the trans face cisternae of the Golgi apparatus (GA). The vesicles apparently move from the GA, found in cytoplasmic depressions between lobes of the plastid, to the sub-plasma membrane peripheral cytoplasm. Here, they become associated with components of the peripheral cytoskeletal network. The mucilage is ultimately released through flask-shaped pores in the cell wall.     

CALCIFICATION IN THE GREEN ALGA HALIMEDA. I. AN ULTRASTRUCTURE STUDY OF THALLUS DEVELOPMENT1

The ultrastructure of 4 species of the calcareous, siphonaceous alga Halimeda (H. cylindracea Decaisne, H. discoidea Decaisne, H. macroloba Decaisne and H. tuna (Ellis & Solander) Lamour) has been studied, and the observed changes during growth and development are related to changes in the degree of calcification. A distinct gradient in the types and quantities of cell organelles exists in a growing apical filament. As these filaments grow, branch, and eventually develop into a mature segment, changes in the organization of organelles such as mitochondria and chloroplasts are observed. Calcification begins when the chloroplasts reach structural maturity and when the peripheral utricles adhere (fuse). This adhesion of the peripheral utricles isolates the intercellular space (ICS) in which calcification occurs from the external seawater. Calcification begins in the outermost (pilose) cell wall layer of the walls facing into the ICS. The cell walls at the thallus exterior undergo extensive changes after utricular fusion; the pilose layer is lost, the cuticles of adjacent utricles fuse forming a ridge at their junction, and multiple cuticles are formed. The aragonite (CaCO₃) crystals which are initially precipitated within the pilose wall layer, rapidly increase in size and number, eventually filling much of the ICS. Only the initial nucleation of aragonite is associated with the pilose wall layer, the later precipitation of aragonite is totally independent of the pilose layer. In older segments secondary deposition of CaCO₃ also occurs around existing aragonite needles.     

PURIFICATION AND CHARACTERIZATION OF TWO FORMS OF PHOSPHOGLYCERATE KINASE FROM THE GREEN ALGA SELENASTRUM MINUTUM1

We report the first purification and characterization of a eukaryotic algal phosphoglycerate kinase (PGK), Two forms of PGK (PGK₁ and PGK₂) from the green alga Selenastrum minutum (Naeg.) Collins were purified to electrophoretic homogeneity with specific activities of 1100 and 1069 units · mg^?1 protein, respectively. The portion of PGK₁ and PGK₂ (probably the cytosolic and chloroplastic forms, respectively) in this organism was estimated as 32 and 68%, respectively. PGK₁ was more heat-stable than PGK₂. The M_r estimation for PGK₁ and PGK₂ by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and gel filtration indicated that they both were monomeric with a similar M_r of approximately 44 kDa. Antibodies raised against S. minutum PGK₁ cross-reacted with PGK₂ as well as PGKs from prokaryotic and eukaryotic sources, suggesting that PGK₁ was structurally and immunologically closely related to PGK₂ and other PGKs, which was consistent with NH₂-terminal sequence analysis. Comparative kinetic and regulatory properties of PGK₁ and PGK₂ from S. minutum were investigated, Both forms exhibited hyperbolic kinetics with respect to both 3-phosphoglycerate (3-PGA) and Mg-adenosine triphosphate^2- (MgATP^2-) under the conditions tested and had similar K_m values for each substrate (PGK₁; K_m (MgATP_2-) = 0.37 mM, K_m(3-PGA) = 0.59 mM; PGK₂; K_m(MgATP^2-) = 0.32 mM, K_m(3-PGA) = 0.46 mM). PGK₁ and PGK₂, however, differed significantly in several other kinetic properties. PGK₂ had a broad pH optimum between 7.3 and 7.8, as compared to PGK₁, with a pH optimum of 7.3 Mg²⁺ was the most efficient cofactor for both forms; it inhibited PGK₁ but not PGK₂ at higher concentrations (>10 mM). Other divalent cations (Mn²⁺, Zn²⁺, Co²⁺, Cd²⁺, and Ca²⁺) only partially replaced Mg²⁺ and were more effective for PGK₁ than for PGK₂, A wide range of metabolites was examined for regulatory properties. Energy charge was the most important factor in regulating the two forms of S. minutum PGK. These results were interpreted in light of the regulation of this kinase in response to the cell energy requirement and the need for glycolytic carbon flow to provide carbon skeletons for amino acid biosynthesis.     

FATTY ACID AND LIPID COMPOSITION OF THE ACIDOPHILIC GREEN ALGA CHLAMYDOMONAS SP.1

The lipid and fatty acid compositions of Chlamydomonas sp. isolated from a volcanic acidic lake and C. reinhardtii were compared, and the effects of pH of the medium on lipid and fatty acid components of Chlamydomonas sp. were studied. The fatty acids in polar lipids from Chlamydomonas sp. were more saturated than those of C. reinhardtii. The relative percentage of triacylglycerol to the total lipid content in Chlamydomonas sp. grown in medium at pH 1 was higher than that in other cells grown at higher pH. A probable explanation might be that Chlamydomonas sp. has two low pH adaptation mechanisms. One mechanism is the saturation of fatty acids in membrane lipids to decrease membrane lipid fluidity, and the other is the accumulation of triacylglycerol, as a storage lipid, to prevent the osmotic imbalance caused by high concentrations of H₂SO₄.     

BIOCHEMICAL, PHYSIOLOGICAL, AND MOLECULAR ANALYSIS OF SEXUAL ISOLATION IN THE SPECIES COMPLEX CLOSTERIUM PERACEROSUM-STRIGOSUM-LITTORALE (CHLOROPHYTA)1

Closterium strains obtained from Japan ( NIES-64 and -65 ) and Nepal ( NIES-67 and -68 ) have been classified as the same taxonomic species; however, they are sexually isolated from each other. When NIES-64 and -65 cells were separately incubated in a medium in which both strains had previously been cultured together, release of protoplasts from both strains was observed. We suggest that factors responsible for the release of protoplasts from cells of both NIES-64 and -65 are produced in a mixed culture of these cells and function during conjugation. These factors, however, had no effect on the release of protoplasts from cells of strains NIFS-67 or -68. Alternatively, a protein that is responsible for the release of protoplasts from cells of NIES-68, called the protoplast-release-inducing protein ( PR-IP ), had no effect on the release of protoplasts from cells of strains NIES-64 or -65. When the media obtained from the culture of NIES-64 and -65 cells at various mixing ratios were analyzed by western blotting with antiserum to a 42-kDa subunit of PR-IP, no cross reaction was detected. In Southern hybridization analysis, no hybridizing band was observed when genomic DNAs of NIES-64 and -65 cells were probed with cDNAs encoding the two subunits of PR-IP. We suggest from these results that the factors responsible for the release of protoplasts from NIES-64 and -65 cells are not structurally similar to PR-IP. It is known that the release of PR-IP from NIES-67 cells can be induced by the action of another sex pheromone ( PR-IP inducer ) which is released by NIES-68 cells. In contrast, no protoplast-release-inducing activity was observed from either NIES-64 or -65 in a culture medium conditioned by opposite strains. We suggest that the conjugation systems employed by strains NIES-64/ NIES-65 and strains NIES-67 /NIES-68 differ, and we propose a possible mechanism of sexual isolation between these biological species .     

COMPARATIVE CYTOLOGY AND TAXONOMY OF THE ULVAPHYCEAE. III. THE FLAGELLAR APPARATUSES OF THE ANISOGAMETES OF DERBESIA TENUISSIMA (CHLOROPHYTA)1

The components of the flagellar apparatuses of the male and female gametes of Derbesia tenuissima (De Not.) Crouan are compared with those in other swarmers of green algae. Both the male and female gametes were found to have a cruciate microtubular root system, a non-striated capping plate which connects basal bodies, two electron dense terminal caps which partially cover the proximal end of the basal bodies, and two small system II fibrous roots. Similarities exist between these components and those suggested to be typical of ulvalean swarmers. Based upon these similarities, it is proposed that the Caulerpales be classified in the Ulvaphyceae rather than in the Charophyceae or Chlorophyceae.     

STRUCTURE OF THE ANISOGAMETES OF THE GREEN SIPHON PSEUDOBRYOPSIS SP. (CHLOROPHYTA)1

The fine structure of the male and female gametes of Pseudobryopsis, particularly that of the flagellar apparatus, is compared with that of swarmers of other green algae. There is general similarity, with differences in detail, to the Ulvales and other green siphons that have been studied. The similarities include overlapping basal bodies, the capping plate type of connective between basal bodies, terminal caps, and system II fibrous roots (rhizoplasts). The capping plate of the female gamete differs from that in other green siphons and the Ulvales in form and in the presence of a faint striation. A diagram illustrating the actual arrangement of the components of the flagellar apparatus is given, along with a discussion of the fact that the mirror image of the true arrangement has been given in some reports on ulvaphycean algae.     

ANALYSIS OF MUCILAGE SECRETION AND EXCRETION IN MICRASTERIAS (CHLOROPHYTA) BY MEANS OF IMMUNOELECTRON MICROSCOPY AND DIGITAL TIME LAPSE VIDEO MICROSCOPY1

Two different, independent, and alternative modes of mucilage excretion were found in the unicellular green alga Micrasterias denticulata Bréb. under constant culture conditions. The cells were capable of either excreting mucilage over all their cell surface or they extruded mucilage from one of their polar ends, which enabled directed movement such as photoorientation or escape from unfavorable environmental conditions. By means of a polyclonal antibody raised against Micrasterias mucilage, the secretory pathway of Golgi derived mucilage vesicles from their origin to their discharge was analyzed by means of conventional and energy filtering TEM. Depending on the stage of the cell cycle, mucilage vesicles were subjected to maturation processes. This may occur either after they have been pinched off from the dictyosomes (e.g. during cell growth) or when still connected to trans‐Golgi cisternae, as in the case of interphase cells. Only fully grown mature vesicles contained mucilage in its final composition as indicated by antibody labeling. After fusion of mucilage vesicles with vacuoles, no immunolabeling was found in vacuoles, indicating that the vesicle content was digested. Mucilage vesicles fused with the plasma membrane in areas of cell wall pores but were also able to excrete mucilage at any site directly through the respective cell wall layer. This result disproves earlier assumptions that the pore apparatus in desmids are the only mucilage excreting areas at the cell surface. Both mechanisms, excretion through the pores and through the cell wall, lead to formation of mucilage envelopes covering the entire cell surface.     

STIMULATION OF NITRATE NET UPTAKE AND REDUCTION BY RED AND BLUE LIGHT AND REVERSION BY FAR-RED LIGHT IN THE GREEN ALGA ULVA RIGIDA1

The steady-state levels of nitrate, nitrite, and ammonium were estimated in the green alga Ulva rigida C. Agardh in darkness after addition of 0.5 mM KNO₃ and irradiation with red (R) and blue (B) light pulses of different duration (5 and 30 min). The net uptake of nitrate was very rapid. Seventy-five percent of the nitrate added was consumed after 60 min in darkness. Although uptake was stable after R or B, efflux of nitrate occurred within 3 h in the dark control and when R or B were followed by far-red (FR) irradiation. The internal nitrate concentration after 3 h in darkness was similar after R and B light pulses; however, the intracellular ammonium was higher after R than after B. The intracellular nitrate and ammonium decreased when FR tight pulses were applied immediately after R or B. Thus, the involvement of phytochrome in the transport of nitrate and ammonium is proposed. Nitrate reductase activity, measured by the in situ method, was increased by both R and B light pulses. The effect was partially reversed by FR light. Nitrate reductase activity was higher after 5 min of R light than after 5 min of B. However, after 30-min light pulses, the relative increase in activity was reversed for R and B. We propose that phytochrome and a blue-light photoreceptor are involved in regulation of nitrogen metabolism. Nitrate uptake and reduction correlates with previously detected light-regulated accumulation of protein in Ulva rigida under the same experimental conditions.     

A STUDY OF CELL WALL AND FLAGELLA FORMATION DURING CELL DIVISION IN THE SCALY GREEN ALGA,SCHERFFELIA DUBIA (CHLOROPHYTA)1

The thecate green flagellate Scherffelia dubia (Perty) Pascher divides within the parental cell wall into two progeny cells. It sheds all four flagella before cell division, and the maturing progeny cells regenerate new walls and flagella. By synchronizing cell division, we observed mitosis, cytokinesis, cell maturation, flagella extension, and cell wall formation via differential interference contrast microscopy of live cells and serial thin‐section EM. Synthesis of thecal and flagellar scales is spatially and temporally strictly separated. Flagellar scales are collected in a pool during late interphase. Before prophase, Golgi stacks divide, flagella are shed, the parental theca separates from the plasma membrane, and flagellar scales are deposited on the plasma membrane near the flagellar bases. At prophase, Golgi bodies start to synthesize thecal scales, continuing into interphase after cytokinesis. During cytokinesis, vesicles containing thecal scales coalesce near the cell posterior, forming a cleavage furrow that is initially oriented slightly diagonal to the longitudinal cell axis but later becomes transverse. After the progeny nuclei have moved into opposite directions, resulting in a “head to tail” orientation of the progeny cells, theca biogenesis is completed and flagellar scale synthesis resumes. Progeny cells emerge through a hole near the posterior end of the parental theca with four flagella of about 8 μm long. The precise timing of flagellar and thecal scale synthesis appears to be an evolutionary adaptation in a scaly green flagellate for the thecal condition, necessary for the evolution of the phycoplast and thus multicellularity in the Chlorophyta.     

PLASMOLYSIS,HECHTIAN STRAND FORMATION,AND LOCALIZED MEMBRANE‐WALL ADHESIONS IN THE DESMID,CLOSTERIUM ACEROSUM (CHLOROPHYTA)1

Closterium acerosum Ehrenberg (Chlorophyta) produced a distinct network of thin cytoplasmic strands, or Hechtian strands, upon controlled plasmolysis in a sucrose solution. The strands persisted for 30 min or longer and could be visualized with both LM and EM. Near the plasma membrane of the polar zones of plasmolyzing protoplasts, the strands formed a “lattice”‐like arrangement with interstrand spacing of 120–130 nm. The strands terminated at the fibrous zone of the inner cell wall stratum. Although actin cables could be found attached to the plasma membrane upon rhodamine phalloidin labeling of membrane ghosts, neither microfilaments nor microtubules were found in Hechtian strands at any stage of development. The formation of strands was not disrupted by centrifugation at 8000 g or by repeated cycles of plasmolysis‐deplasmolysis. Application of microtubule‐ or microfilament‐affecting agents or various proteolytic/polysaccharide‐degrading enzymes did not disrupt the formation of strands. Cold treatment of cells resulted in the formation of Hechtian strands.     

GROWTH AND POLYSACCHARIDE PRODUCTION BY THE GREEN ALGA CHLAMYDOMONAS MEXICANA (CHLOROPHYCEAE) ON SOIL1

The polysaccharide producing soil alga Chlamydomonas mexicana Lewin was grown on soil surfaces in a growth chamber, with cell number and total polysaccharide measured weekly. Cell growth was pronounced, reaching an excess of 10⁷ cells · cm^?2 within one week. Polysaccharide production was also pronounced, with similar amounts of polysaccharide synthesized whether or not the cells were continuously given nutrients. Polysaccharide synthesis increased once the cells slowed in their growth, as in previous work with the cells in liquid medium.     

STRUCTURE OF THE CAPSULAR AND EXTRACELLULAR POLYSACCHARIDES PRODUCED BY THE DESMID SPONDYLOSIUM PANDURIFORME (CHLOROPHYTA)1

The filamentous desmid Spondylosium panduriforme (Heimerl) Teiling var. panduriforme f. limneticum (West & West) Teiling (Desmidiaceae), strain 072CH-UFCAR, is surrounded by a well-defined, mucilaginous capsule consisting of a capsular polysaccharide (CPS). This microalga also produces an extracellular polysaccharide (EPS), which can be isolated from the culture medium. Analysis of the carbohydrate composition of the two polymers by gas chromatography showed that they were different. Both were composed, of galactose, fucose, xylose, arabinose, rhamnose, and glucose but in different amounts. For example, glucuronic acid accounts for 24% of the EPS material but only traces were found in the CPS. Significant differences were also found during methylation analysis. Fucose appeared to have a higher degree of branching in the EPS than in the CPS. These branches were located on C-3 and could be the position for the attachment of the glucuronic acid units in the EPS. The glucuronic acid was present as 1→4-linked and terminal units. A possible explanation for the formation of the EPS is suggested.     

GROWTH OF THE FILAMENTOUS GREEN ALGA CTENOCLADUS CIRCINNATUS (CHAETOPHORALES,CHLOROPHYCEAE) IN RELATION TO ENVIRONMENTAL SALINITY1

Clones of the filamentous green alga Ctenocladus circinnatus Borzi were isolated from algae collected at Abert Lake (Oregon) and Mono Lake (California). Stock cultures were exposed to varied salinities of natural lake water to examine the effects on growth rate, cell form, chlorophyll a, and water content. Growth rates were reduced in both clones with increased salinity over the range 25–100 g·L^?1 and were almost completely inhibited at 150 g·L^?1. Chlorophyll a increased between salinities of 25 and 100 g·L^?1, reflecting slower growth, higher proportions of akinetes, and smaller cell sizes as salinity increased. Tissue water content remained essentially constant from 25 to 100 g·L^?1 salinity. Shorter cell dimensions with increased salinity suggest that a lower surface-to-volume ratio may reduce the potential for passive loss of cell water. Prior acclimation of stock cultures to elevated salinity provided no enhancement of growth response at any salinity. The results indicate that environmental salinity can limit the productivity and distribution of Ctenocladus in nature.     

CELL DIVISION AND COLONY FORMATION IN THE GREEN ALGA COELASTRUM (CHLOROCOCCALES)1

Multinucleate cells of Coelastrum undergo precisely directed cytokinesis, guided by phycoplast microtubules, to form a number of uninucleate daughter cells which subsequently adhere to form characteristically patterned aggregates. As there is no movement of the daughter cells relative to one another before their adhesion, the disposition of cells in daughter colonies reflects the pattern of cytokinesis of parent cells. Centrioles lie at the poles of the mitotic nuclei which are partially enclosed by a perinuclear envelope of endoplasmic reticulum. The centrioles disappear at the time of cytokinesis of the parental cell and apparently reform de novo once the daughter cells have acquired a cell wall following their adhesion. The trilaminar layer of cell wall, often termed the pectic layer, does not stain with ruthenium red and resists acetolysis suggesting that it contains sporopollenin rather than pectin.     

SEXUAL PROCESSES AND PHYLOGENETIC RELATIONSHIPS OF A HOMOTHALLIC STRAIN IN THE CLOSTERIUM PERACEROSUM–STRIGOSUM–LITTORALE COMPLEX (ZYGNEMATALES,CHAROPHYCEAE)1

Members of the Closterium peracerosum–strigosum–littorale (C. psl.) complex are unicellular charophycean algae in which there are two modes of zygospore formation, heterothallic and homothallic. A homothallic strain of Closterium (designation, kodama20) was isolated from a Japanese rice paddy field. Based on alignment of the 1506 group‐I introns, which interrupt nuclear SSU rDNAs, homothallic kodama20 is most closely related to the heterothallic mating group II‐B, which is partially sexually isolated from group II‐A. Time‐lapse photography of the conjugation process in kodama20 revealed that most of the observed zygospores originated from one vegetative cell. The sexual conjugation process consisted of five stages: (1) cell division resulting in the formation of two sister gametangial cells from one vegetative cell, (2) formation of a sexual pair between the two sister gametangial cells (or between gametangial cells of another adjoined individual), (3) formation of conjugation papillae, (4) release of gametic protoplasts from both members of a pair, and (5) formation of the zygospore by protoplast fusion. For conjugation to progress, the cell density and light condition in the culture was critical. We suggested the presence of a conjugation promotion factor.     

MICROSENSOR STUDIES OF OXYGEN AND LIGHT DISTRIBUTION IN THE GREEN MACROALGA CODIUM FRAGILE1

Scalar irradiance, oxygen concentration, and oxygenic photosynthesis were measured at 0.1 mm spatial resolution within the tissue of the siphonous green macroalga Codium fragile subsp. tomentosoides (van Goor) Silva by fiber-optic scalar irradiance microsensors and oxygen microelectrodes. The scalar irradiance of visible light was strongly attenuated in the outer 0.2 mm of the tissue but was nearly constant for the subsequent 1.0 mm of photo-synthetic tissue. Far-red scalar irradiance at 750 nm increased below the tissue surface to a maximum of 200% of incident irradiance at 1.2 mm depth due to multiple scattering in the medullary tissue. The constant intensity of visible light below 0.2 mm was thus a result of the combined effects of absorption and backscattering from the medulla. The oxygen exchange between the alga and the surrounding water was diffusion-limited with a steep O₂gradient inside and around the alga. In darkness, the tissue below 0.6 mm became anoxic, and endophytic extracellular space provided an environment where anoxygenic microbial processes may occur. When illuminated at 160 nmol photons·^?2·^?1, O₂ concentrations exceeded ambient levels throughout the thallus, with a maximum of 250% of air saturation just below the surface. The amplitude of oxygen variation was buffered by gas bubbles formed in the medullary tissue.     

CONCORDANCE OF MOLECULAR AND ULTRASTRUCTURAL DATA IN THE STUDY OF ZOOSPORIC CHLOROCOCCALEAN GREEN ALGAE1

Alternative evolutionary hypotheses generated from features of vegetative cell morphology and motile cell ultra-structure were investigated using a molecular data set. Complete nuclear-encoded small subunit (18S) ribosomal RNA (rRNA) gene sequences were determined for six species (three each) of the chlorococcalean green algae “Neo chloris” and Characium. Based on motile cell ultra-structure, it was previously shown that both genera could be separated into three distinct groups possibly representing three separate orders and two classes of green algae. 18S rRNA gene sequences were also obtained for three additional taxa, Dunaliella parva Lerche, Pediastrum duplex Meyen, and Friedmannia israelensis Chantanachat and Bold. These organisms were selected because each, in turn, is a representative of one of the three ultrastructural groups into which the Neochloris and Characium species are separable. Phylogenetic analyses utilizing the molecular data fully support the ultrastructural findings, suggesting that the similar vegetative cell morphologies observed in these organisms have resulted from convergence.     

THE FOSSIL GREEN ALGA MIZZIA (DASYCLADACEAE): A TOOL FOR INTERPRETATION OF PALEOENVIRONMENT IN THE UPPER PERMIAN CAPITAN REEF COMPLEX SOUTHEASTERN NEW MEXICO1

A paleoenvironmental study of the dasyclad Mizzia Schubert, 1907 is potentially important because, unlike most of the other fossil reef-dwelling organisms found in the Guadalupe Mountains, the fossil Mizzia (restricted to the Permian) has a modern analog, Cymopolia Lamouroux, 1916 (Cretaceous to Holocene). The overall morphology of the two genera is similar: both are articulated, and observations on our specimens suggest that both were branched. The transition from the remarkably high-diversity sponge and algal assemblage in the reef into the remarkably low-diversity dasyclad-dominated assemblage in the back reef is abrupt, occurring over distances as short as 10 m. Mizzia is the dominant and usually the only component in grainstones, packstones, and wackestones found; immediately shelfward of the Capitan reef. The low percentage of broken segments (average 10%) and the discovery of several intact sections of thallus are evidence against wave action as a possible limiting factor. Because the climate in the Guadalupe Mountains was extremely arid and because dasyclads are euryhaline, hypersalinity is considered the most likely limiting factor in this case. Our conclusion that prolific stands of dasyclads formed in shallow protected lagoons immediately behind an emergent barrier reef is not compatible with the currently accepted marginal mound hypothesis for deposition of the Capitan reef complex. We suggest that the marginal mound model must either be modified or abandoned in favor of the originally proposed barrier reef model of deposition.     

CELL WALL STRUCTURE AND IRON DISTRIBUTION IN THE GREEN ALGA STAURASTRUM LUETKEMUELLERI (DESMIDIACEAE)1

The cell wall of Staurastrum luetkemuelleri Donnat & Ruttner was examined with scanning electron microscope (SEM) using whole cells, in thin sections with transmission electron microscope (TEM), and in air dried whole cells and unstained thin sections with X-ray microanalysis in the scanning-transmission electron microscope (STEM). The cell wall was ornamented with spines and wartlike structures. Spines were solid structures, consisting of deposits of cell wall material between two main cell wall layers. The wart-like structures were pore organs extending through the cell wall and the mucilaginous layer outside the cell wall. The pore cylinder was surrounded by deposits of cell wall material similar to the ones in the spines. X-ray microanalysis of selected areas on whole cells from a natural population showed iron accumulation in discrete locations on the cell extensions of S. luetkemuelleri. In the unstained thin sections iron was found only in the cell wall deposits in the spines. Cells grown in laboratory cultures failed to show iron accumulation regardless of readdition of iron-EDTA (Fe-EDTA) to the culture medium.