ULTRASTRUCTURE OF A CHAIN-FORMING DIATOM PHAEODACTYLUM TRICORNUTUM1

The ultrastructure of a chain-forming clone of the polymorphic diatom Phaeodactylum tricornutum Bohlin has been studied by scanning and transmission electron microscopy. Both fusiform and tri-radiate cells are capable of forming chains. The cells, lacking any silica shell, are attached to each other at the central region of the theca, leaving the arms free. Neither homogenization nor sonication completely disrupts the chains. The attachment is due to fusion of the cell wall in the central region of the cell during cell wall deposition. This fusion results from failure of the cytoplasmic cleavage furrow to separate the plasma membranes of the two daughter cells sufficiently so that a single wall is deposited instead of two separate walls. Possible explanations for this are discussed.     

ULTRASTRUCTURE OF THE FRUSTULE OF TRICERATIUM FAVUS (BACILLARIOPHYCEAE)1,2

New structural details of the frustules of the diatom Triceratium favus Ehrenberg seen in the scanning electron microscope are reported. Significant new observations concern the pores of the hexagonal chambers, accessory structures (spines, dendritic processes) on the outer surfaces of the hexagonal chambers, the value margins and girdle structure.     

UPTAKE OF GUANINE BY THE DIATOM,PHAEODACTYLUM TRICORNUTUM1

Nitrate-cultured cells of Phaeodactylum tricornutum Bohlin lack the ability to take up guanine but can do so after a period of nitrogen deprivation, i.e. photosynthesis in nitrogen-free medium. Maximum rate of uptake occurred after 24 h of nitrogen deprivation. The development of ability to take up guanine required CO₂ fixation and was prevented by cycloheximide, ammonium or nitrate. The guanine taken up accummulated in the cells almost entirely as a compound which is probably methylated hypoxanthine. Guanine uptake was dependent upon metabolism and exhibited Michaelis-Menten like kinetics with a half-saturation value of 0.48 ± 0.05 μM guanine and a maximum uptake rate for guanine of ca. 200 nmol · 10^?8 cells · h^?1. Rate of uptake increased hyperbolically with Na⁺ concentration, with 8.25 mM Na⁺ supporting half-maximal rate, and it was inhibited by K⁺ ions.     

PROTEINACEOUS AND IMMUNOCHEMICAL DISTINCTIONS BETWEEN THE OVAL AND FUSIFORM MORPHOTYPES OF PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Two morphotypes, fusiform and oval, were isolated from a single clone of the diatom Phaeodactylum tricornutum Bohlin and maintained as subclones by culturing in liquid and solid substrates, respectively. Salinity of the medium, from brackish to marine, had no effect on expression of the phenotypes. The oval cell is generated endogenously within a “transformed”fusiform cell upon transfer from liquid medium to agar plates. With the light microscope, normal and “transformed”fusiform cells, prior to giving rise to oval cells, can be discriminated by means of their staining response to toluidine blue. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of protein extracts from lysed cells revealed slight differences in polypeptide composition between fusiform and oval types. A phenotype-restoration experiment from oval to fusiform demonstrated that the oval type readily reestablished not only fusiform morphology but also the protein pattern characteristic for the fusiform type. Immunochemical analyses (western blots) using antisera raised against whole and lysed cells of both morphotypes revealed antigenic alterations of the oval morphotype. Several antigenic determinants restricted mainly to the surface of oval cells were detected. Results indicate that environmentally induced phenotypes of Phaeodactylum may be not only the consequence of specific gene expression but also the result of significant, general post-translational modifications.     

解淀粉芽孢杆菌对三角褐指藻的化感作用研究

实验研究不同剂量(100、500和1000μL)的解淀粉芽孢杆菌菌液、解淀粉芽孢杆菌代谢产物和解淀粉芽孢杆菌(Bacillus amyloliquefaciens)及代谢产物混合液3种组合对三角褐指藻(Phaeodactylum tricornutum)生长的影响.结果表明,解淀粉芽孢杆菌、其代谢产物和两种的混合液对三...     

CHANGES IN ABSORBANCE SPECTRUM OF THE DIATOM PHAEODACTYLUM TRICORNUTUM UPON MODIFICATION OF PROTEIN STRUCTURE1,2

Changes in the absorbance spectrum, of the diatom Phaeodactylum tricornutum which are brought about by heat, urea, stilt, and formaldehyde have been studied. The changes brought about by all agents are similar. Each converts cell suspensions from orange-brown to yellow-green. This color change results from an increase in absorbance in the blue and a decrease in absorbance in the green. All agents, except salt, also bring about an increase in absorbance in the red at wavelengths below that of the red peak of chlorophyll, and a decrease in absorbance at longer wavelengths. On heating cells, the absorbance change in the green occurs more rapidly than the changes in the blue and red. The converse is true when cells are treated with 4 M NaCl. Heat or formaldehyde treatments chemically modify chlorophyll pigments which are present in untreated cells. The change in absorbance in the green is probably due to changes in the molecular environment of fucoxanthin which, results from protein modification. The changes in the blue and red are at least partly due to chemical changes in chlorophylls, but may also be due, in part, to protein modification.     

VARIATION IN THE OCCURRENCE OF EXTERNAL CARBONIC ANHYDRASE AMONG STRAINS OF THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Eleven different strains of Phaeodactylum tricornutum Bohlin were obtained from three culture collections and were examined for the presence of external and internal carbonic anhydrase (CA). Cells of all strains, grown in standing culture at alkaline pH and low, dissolved inorganic carbon had internal CA, but only eight were found to have external CA. External CA activity was reduced when cultures were bubbled with air and was completely repressed when they were grown on 5% CO₂. Expression of external CA activity appears to be regulated by CO₂ concentration in the growth medium, but within one species, there appears to be a variation in occurrence of external CA and consequently in the mode of inorganic carbon acquisition.     

EFFECT OF OLIGOMYCIN ON DARK RESPIRATION IN THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE): IMPLICATIONS FOR DETERMINATION OF MAINTENANCE RESPIRATION

Oligomycin is an inhibitor of the mitochondrial ATP synthase. In nitrogen-replete cells of the marine diatom Phaeodactylum tricornutum Bohlin, the rate of dark respiration was high and markedly inhibited (62%–74%) in the presence of oligomycin. In contrast, the rate of dark respiration in nitrogen-deprived cells was about half that in nitrogen-replete cells but was only slightly inhibited (16%–30%) by oligomycin. Consistent with these effects on rates of dark respiration, oligomycin decreased the ATP level and the ATP:ADP ratio by about 40% in nitrogen-replete cells incubated in darkness but had a negligible effect on the ATP level and ATP:ADP ratio in nitrogen-deprived cells. In sodium and nitrogen-deprived cells, the rate of dark respiration was greater than that in nitrogen-replete cells, but there was little effect of oligomycin on the rate of dark respiration. In light-limited cells, the rate of dark respiration was similar to that in nitrogen-deprived cells, but the inhibition (57%) in the presence of oligomycin was greater. These results suggest that most of the O₂ consumption by nitrogen-replete cells was linked to mitochondrial ATP synthesis and that the rate of mitochondrial ATP synthesis in nitrogen-deprived and sodium and nitrogen-deprived cells was low. The potential implications of these results for our understanding of maintenance respiration are discussed.     

COMBINED EFFECTS OF TEMPERATURE AND IRON ON THE GROWTH AND PHYSIOLOGY OF THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)

Phaeodactylum tricornutum Bohlin (Bacillariophyceae) was maintained in exponential growth under Fe‐replete and stressed conditions over a range of temperatures from 5 to 30° C. The maximum growth rate (GR) was observed at 20° C (optimal temperature) for Fe‐replete and ‐stressed cells. There was a gradual decrease in the GR decreasing temperatures below the optimum temperature; however, the growth rate dropped sharply as temperature increased above the optimum temperature. Fe‐stressed cells grew at half the growth rate of Fe‐replete cells at 20° C, whereas this difference became larger at lower temperatures. The change in metabolic activities showed a similar pattern to the change in growth rate temperature aside from their optimum temperature. Nitrate reductase activity (NRA) and respiratory electron transport system activity (ETS) per cell were maximal between 15 and 20° C, whereas cell‐specific photosynthetic rate (P_cell) was maximal at 20° C for Fe‐replete cells. These metabolic activities were influenced by Fe deficiency, which is consistent with the theoretical prediction that these activities should have an Fe dependency. The degree of influence of Fe deficiency, however, was different for the four metabolic activities studied: NRA > P_cell > ETS = GR. NRA in Fe‐stressed cells was only 10% of that in Fe‐replete cells at the same temperature. These results suggest that cells would have different Fe requirements for each metabolic pathway or that the priority of Fe supply to each metabolic reaction is related to Fe nutrition. In contrast, the order of influence of decreasing the temperature from the optimum temperature was ETS > P_cell > NRA > GR. For NRA, the observed temperature dependency could not be accounted for by the temperature dependency of the enzyme reaction rate itself that was almost constant with temperature, suggesting that production of the enzyme would be temperature dependent. For ETS, both the enzyme reactivity and the amount of enzyme accounted for the dependency. This is the first report to demonstrate the combined effects of Fe and temperature on three important metabolic activities (NRA, P_cell, and ETS) and to determine which activity is affected the most by a shortage of Fe. Cellular composition was also influenced by Fe deficiency, showing lower chl a content in the Fe‐stressed cells. Chl a per cell volume decreased by 30% as temperature decreased from 20 to 10° C under Fe‐replete conditions, but chl a decreased by 50% from Fe‐replete to Fe‐stressed conditions.     

EXTRACELLULAR MATRIX ASSEMBLY IN DIATOMS (BACILLARIOPHYCEAE). V. ENVIRONMENTAL EFFECTS ON POLYSACCHARIDE SYNTHESIS IN THE MODEL DIATOM,PHAEODACTYLUM TRICORNUTUM1

The effects of phosphate (P) limitation, varying salinity (5–65 psu), and solid media growth conditions on the polysaccharides produced by the model diatom, Phaeodactylum tricornutum Bohlin were determined. Sequential extraction was used to separate polymers into colloidal (CL), colloidal extracellular polymeric substances (cEPS), hot water soluble (HW), hot bicarbonate soluble (HB), and hot alkali (HA) soluble fractions. Media‐soluble polymers (CL and cEPS) were enriched in 4‐linked mannosyl, glucosyl, and galactosyl residues as well as terminal and 3‐linked xylosyl residues, whereas HW polymers consisted mainly of 3‐linked glucosyl as well as terminal and 2,4‐linked glucuronosyl residues. The HB fraction was enriched in terminal and 2‐linked rhamnosyl residues derived from the mucilage coating solubilized by this treatment. Hot alkali treatment resulted in the complete dissolution of the frustule releasing 2,3‐ and 3‐linked mannosyl residues. The fusiform morphotype predominated in standard and P‐limited cultures and cultures subjected to salinity variations, but growth on solid media resulted in an enrichment of the oval morphotype. The proportion and linkages of 15 residues, including neutral, uronic acid, and O‐methylated sugars, varied with environmental conditions. P limitation and salinity changes resulted in 1.5‐ to 2.5–fold increase in carbohydrate production, with enrichment of highly branched/substituted and terminal rhamnose, xylose, and fucose as well as O‐methylated sugars, uronic acids, and sulfate. The increased deoxy‐ and O‐methylated sugar content under unfavorable environments enhances the hydrophobicity of the polymers, whereas the anionic components may play important roles in ionic cross‐linking, suggesting that these changes could ameliorate the effects of salinity or P‐stress and that these altered polysaccharide characteristics may be useful as bioindicators for environmental stress.     

EFFECTS OF LOWERING TEMPERATURE DURING CULTURE ON THE PRODUCTION OF POLYUNSATURATED FATTY ACIDS IN THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

The composition of fatty acids and contents of eicosapentaenoic acid (EPA) and polyunsaturated fatty acids (PUFAs) of the economically important marine diatom, Phaeodactylum tricornutum (Bohlin), were investigated to see whether reducing the culture temperature enhances the production of EPA and PUFAs. The contents of EPA and PUFAs of P. tricornutum were found to be higher at lower temperature when cultured at 10, 15, 20, or 25°C. When the cells grown at 25°C were shifted to 20, 15, or 10°C, the contents per dry mass of PUFAs and EPA increased to the maximal values in 48, 24, and 12 h, respectively. The highest yields of PUFAs and EPA per unit dry mass (per unit volume of culture) were 4.9% and 2.6% (12.4 and 6.6 mg·L^?1), respectively, when temperature was shifted from 25 to 10°C for 12 h, both being raised by 120% compared with the control. The representative fatty acids in the total fatty acids, when temperature was lowered from 25 to 10°C, decreased proportionally by about 30% in C_16:0 and 20% in C_16:1(n?7) but increased about 85% in EPA. It was concluded that lowering culture temperature of P. tricornutum could significantly raise the yields of EPA and PUFAs.     

INTRACELLULAR AND EXTRACELLULAR AMINO ACID POOLS OF THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) GROWN ON UNENRICHED SEAWATER IN HIGH-CELL-DENSITY DIALYSIS CULTURE1

The consumption of inorganic macronutrients (NO₃^?+ NO₂^?, NH₄⁺, and PO₄^?3) and the composition of intra- and extracellular dissolved free amino acid pools (IDFAA and EDFAA, respectively) were determined in continuous-reservoir batch dialysis cultures of the marine diatom Phaeodactylum tricornutum Bohlin maintained on unenriched natural seawater as a growth medium. Nutrient diffusion (Nd), which equals the nutrient uptake of the culture, increased with the cell density and the age of the culture. A concentration of 6.77 × 10⁷ cells · mL^?1 was obtained in stationary phase, which coincided with the NO₃^?+ NO₂^? diffusion limit (Nd_max) of the dialysis apparatus. The Nd_max for NH₄⁺ occurred much earlier, at the end of exponential growth, whereas Nd_max for PO₄^?3 was not attained during the growth cycle of the culture, even in early stationary phase. A significant depletion (77%) of the IDFAA pool during exponential phase was followed by a reestablishment–to approximately 60% of the initial level–of internal pools during linear and stationary growth phases. This recovery occurred during the illuminated portion of the photoperiod (12:12 h LD) and involved principally the amino acids GLN, GLU, β-GLU, and ASN. The recovery of GLN and ASN levels was particularly significant, because the intracellular concentrations of these amino acids were higher at the end of the growth cycle than before. The EDFAA pool was generally dominated by the amino acids SER and GLY+THR; however, during active growth, ORN and LYS often constituted an important fraction. The EDFAA concentration increased until linear growth phase was reached, during which a higher concentration of total free amino acids was attained in darkness than under illumination. The EDFAA component diminished afterward, and in stationary phase this fraction returned to concentrations equivalent to those observed at the beginning of the growth cycle. The variations in EDFAA concentrations were expressed by a pronounced decrease in the cellular excretion of amino acids with increasing cell density. These cellular responses of Phaeodactylum tricornutum in dense culture, specifically the regulation of amino acid excretion and intracellular pool size, may affect the N-conversion coefficient (Y_N). Consequently, by prolonging the linear phase of growth and reducing the concentration of autoinhibitory metabolites by diffusion, a markedly enhanced final cell density can be achieved in cultures grown on natural unenriched seawater.     

CHANGES IN CELL COMPOSITION AND LIPID METABOLISM MEDIATED BY SODIUM AND NITROGEN AVAILABILITY IN THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

The effects of nitrogen starvation in the presence or absence of sodium in the culture medium were monitored in batch cultures of the marine diatom Phaeodactylum tricornutum Bohlin. During nitrogen starvation in the presence of sodium, cell nitrogen and chlorophyll a decreased, mainly as a consequence of continued cell division. These decreases were accompanied by decreases in the rates of photosynthesis and respiration. There was no change in either cell volume or carbohydrate, but both carbon and lipid increased. During nitrogen starvation in the absence of sodium, cell division ceased. Cell nitrogen and chlorophyll a remained constant, and respiration did not decrease, but the changes in the photosynthetic rate and the lipid content per cell were similar to cultures that were nitrogen-starved in the presence of sodium. The carbon-to-nitrogen ratio increased in both cultures. Nitrogen, in the form of nitrate, and sodium were resupplied to cultures that had been preconditioned in nitrogen- and sodium-deficient medium for 5 d. Control cultures to which neither nitrate or sodium were added remained in a static state with respect to cell number, volume, and carbohydrate but showed slight increases in lipid. Cells in cultures to which 10 mM nitrate alone was added showed a similar response to cultures where no additions were made. Cells in cultures to which 50 mM sodium alone was added divided for 2 d, with concomitant small decreases in all measured constituents. Cell division resumed in cultures to which both sodium and nitrate were added. The lipid content fell dramatically in these cells and was correlated to metabolic oxidation via measured increases in the activity of the glyoxylate cycle enzyme, isocitrate lyase. We conclude that lipids are stored as a function of decreased growth rate and are metabolized to a small extent when cell division resumes. However, much higher rates of metabolism occur if cell division resumes in the presence of a nitrogen source.     

EFFECTS OF SELENIUM DEFICIENCY ON THE MORPHOLOGY AND ULTRASTRUCTURE OF THE COASTAL MARINE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1,2

The effects of selenium deficiency on the siliceous and nonsiliceous components of the planktonic marine diatom Thalassiosira pseudonana (Hust.) Hasle and Heimdal (clone 3H) are examined using light and electron microscopy. Selenium deficiency induces elongation along the pervalvar axis initially as a result of chain formation caused by the failure of sibling cells to separate and subsequently by cell elongation via the production of hyaline girdle bands. In Se-deficient cultures cell elongation involves the blockage of both mitotic and cytokinetic components of cell division. Selenium deficiency results in ultra-structural alterations in the reticular membrane system and in mitochondrial and chloroplast membranes. Various types of inclusions are seen in vacuolar areas and the accumulation of lipid reserves is evident in Se-deficient cells. These results provide indirect evidence for a metabolic Se requirement in this algal species.     

TAXONOMY AND FRUSTULAR STRUCTURE OF THE MARINE CENTRIC DIATOM PARALIA SULCATA1

Light and electron microscopy were used to investigate the complex structure of the frustule of Paralia sulcata (Ehrenb.) Cleve. Rimoportulae are reported for the first time in this diatom and two types of linking processes are described. The ease with which the cingulum is lost is explained with regard to its attachment to the valve. Two kinds of heterovalvy were observed and the taxonomic significance of one of these is discussed. The validity of Heiberg's genus Paralia is confirmed and a type slide of the species is designated.     

EFFECTS OF SELENIUM DEFICIENCY ON THE MORPHOLOGY AND ULTRASTRUCTURE OF THE COASTAL MARINE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1,2

The effects of selenium deficiency on the siliceous and nonsiliceous components of the planktonic marine diatom Thalassiosira pseudonana (Hust.) Hasle and Heimdal (clone 3H) are examined using light and electron microscopy. Selenium deficiency induces elongation along the pervalvar axis initially as a result of chain formation caused by the failure of sibling cells to separate and subsequently by cell elongation via the production of hyaline girdle bands. In Se-deficient cultures cell elongation involves the blockage of both mitotic and cytokinetic components of cell division. Selenium deficiency results in ultrastructural alterations in the reticular membrane system and in mitochondrial and chloroplast membranes. Various types of inclusions are seen in vacuolar areas and the accumulation of lipid reserves is evident in Se-deficient cells. These results provide indirect evidence for a metabolic Se requirement in this algal species.     

EFFECTS OF LIGHT INTENSITY AND TEMPERATURE INTERACTIONS ON GROWTH CHARACTERISTICS OF PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Cell division rate, carbon fixation per cell, cell width and chloroplast length of Phaeodactylum tricornutum Bohlin were determined at 30 different combinations of light intensity and temperature. Division rate peaked at 23° C or less depending on light intensity. For each light intensity studied, carbon fixation increased directly with growth temperature from 14 to 25° C. The slope of this relationship was modified by light intensity. Cells grown at 23–25° C tended to be larger than those grown at lower temperatures, possibly due to increased carbon fixation per cell coupled with lower division rates. Chloroplasts were largest at a combination of temperatures above 21° C and low light intensities. This effect could cause cells to sink at a higher than normal rate due to reduced vacuole size and is presented as a possible mechanism affecting the distribution of P. tricornutum.     

POLYMORPHISM OF THE DIATOM PINNULARIA BREBISSONII IN CULTURE AND A FIELD COLLECTION1,2

Two clones of Pinnularia brebissonii (Kütz.) Rabh. var. brebissonii were established and maintained in logarithmic phase of growth. Initial length of the cells was 37 μm. As cell division occurred, the mean length of cells in each population decreased as predicted by the MacDonald-Pfitzer hypothesis; however, the decrease in mean length was not uniform throughout the growth period. This nonuniformity is probably caused by nonrandom division of cells in the population or by a changing increment of size reduction due to division. The initial increment of size reduction was calculated as 0.7 μm/division. The smallest, cells observed were 8 μm long. As cells decrease in length, cell volume decreases and the proportion of cells with aberrant valve structure increases. More than 90% of the valves were abnormal in a population with mean length of 14 μm. The abnormalities of structure involved the raphe, the central area and the striae.