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.     

QUANTITATIVE GENETICS OF FATTY ACID VARIATION IN ISOCHRYSIS GALBANA (PRYMNESIOPHYCEAE) AND PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Fatty acid variation among culture collection strains and 40 new isolates of Isochrysis galbana Parke was analyzed by quantitative genetic methods. Fatty acid variation among strains and among isolates was highly significant for major fatty acids showing the existence of a genetic component in the determination of differences in fatty acid content. The heritabilities for the major fatty acids ranged between 0.68 and 0.99 among collection strains and between 0.31 and 0.43 among isolates. Eicosapentaenoic acid (EPA) had the highest heritability in I. galbana, but the majority of remaining fatty acids also showed high heritability values. A similar experiment with five UTEX strains of Phaeodactylum tricornutum also showed the presence of a genetic component in four out of seven major fatty acids. Nevertheless, the UTEX strains did not differ significantly in EPA content, although they showed a heritability of 0.40 for this fatty acid. An additional experiment culturing the same isolates of I. galbana in larger volumes of media showed that there was a high significant positive linear relation between EPA content in different volumes. Therefore, EPA content in small volume cultures was an unbiased indicator of EPA content in larger volume cultures. Our results provide support for the genetic determination of fatty acid content in microalgae and suggest that selection, and mutation and selection, are likely to improve EPA content in I. galbana and probably in many other microalgae. Such a selection program can be carried out in small-volume cultures with high confidence.     

CHRONIC EFFECTS OF ULTRAVIOLET-B RADIATION ON GROWTH AND CELL VOLUME OF PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Cultures of the temperate estuarine diatom, Phaeodactylum tricornutum Bohlin (NEPCC Clone 31), were grown under ambient intensities of ultraviolet-A radiation (UVAR), photosynthetically active radiation (PAR), and various intensities of ultraviolet-B radiation (UVBR; 290–320 nm). Growth rates and cell volumes were monitored for 36 d. UVBR decreased growth rates and increased cell volumes. Sensitivity of growth to UVBR increased with time. Growth rates of P. tricornutum decreased with increasing ratios of UVBR:UVAR + PAR.     

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.     

FIRST INSIGHTS INTO IMPROVEMENT OF EICOSAPENTAENOIC ACID CONTENT IN PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) BY INDUCED MUTAGENESIS1

A strain improvement program was initiated based on mutagenesis with the goal of commercial production of eicosapentaenoic acid (EPA)from EPA-overproducing microalgal strains. Two rounds of mutation and selection were conducted using Phaeodactylum tricornutum Bohlin UTEX #640 as the parent strain. After the first round of mutagenesis, a putative mutant (provisionally labeled 114) was obtained. The EPA content (% of dry weight) of this mutant strain was 37% higher than that of the wild type. 114 was further mutated and another putative mutant (provisionally called II242) was isolated, the EPA content of which was 44% higher than that of the wild type. When cultured with aeration in 1-L flasks, EPA content of the wild type and putative mutants 114 and II242 was, 17.3 mg · g^?1, 31.5mg · g^?1, and 38.6 mg · g^?1 dry biomass, respectively. EPA productivity was 3.48 mg · L^?1· d^?1 4.01 mg · L^?1· d^?1, and 4.98 mg · L^?1· d^?1 respectively. These figures compare favorably with many other promising EPA-producing microorganisms and suggest that the use of a single methodology such as mutation and selection is a way to improve the polyunsaturated fatty acid content of microalgae and other microorganisms.     

DEMONSTRATION OF NET INFLUX OF FREE AMINO ACIDS IN PHAEODACTYLUM TRICORNUTUM USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY1

Influx of glycine from dilute solution in the medium into Phaeodactylum tricornutum (Bohlin) was studied using radiochemical techniques. Comparison of these data with simultaneous measurements of the disappearance of primary amines from the medium by fluorometry indicates that influx of ¹⁴C-labeled glycine accurately reflects net entry of substrate. High performance liquid chromatography (HPLC) was employed to demonstrate net removal of fourteen different amino acids from dilute solution by P. tricornutum. HPLC was also used to demonstrate net removal of free amino acids naturally present in sea water.     

EFFECTS OF ENVIRONMENTAL FACTORS ON PHOTOSYNTHESIS PATTERNS IN PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE). I. EFFECT OF NITROGEN DEFICIENCY AND LIGHT INTENSITY1

Cultures of the marine diatom Phaeodactylum tricornutum Bohlin incorporated, a large proportion of the total fixed carbon (50% or more) into amino acids and amides during short periods of photo-assimilation of ¹⁴C-labelled carbon dioxide. Although increasing nitrogen limitation in a nitrate-limited chemostat had little significant effect on the proportion of C incorporated into amino acids and amides combined, it did affect the distribution of radioactivity within individual compounds of this group. In particular, increasing degrees of N deficiency reduced the proportion incorporated into amides to almost undetectable levels, reduced the proportion in alanine and increased the proportion in glutamic acid. Also, increasing N limitation decreased the relative synthesis of sugar phosphates and increased the proportion of C assimilated into intermediates of the tricarboxylic acid cycle. Reduced light intensity did not have any significant effect on the proportion of C incorporated into the total amino acids and amides, but did cause a decrease in the radioactivity     

氮源及其浓度对三角褐指藻生长和脂肪酸组成的影响

为了研究氮源的类型和浓度对微藻脂肪酸组成的影响 ,用含有不同浓度NO3 -、NH4 、NH2 CONH2 的培养基 ,对三角褐指藻 (Phaeodactylumtricornutum)进行了培养 ,并测定了其生长和脂肪酸组成。结果表明 ,培养基中不添加氮源时 ,三角褐指藻生长缓慢 ,但多不饱和脂肪酸 (PUFAs)和C18脂肪酸 (C18∶0 ,C18∶2 (n -6) ,C18∶3 (n -6) )占总脂肪酸的比例较高 ;氮浓度较低 (<1 8mmol/L)时 ,三角褐指藻以NH4 为氮源 ,生长较快 ;氮浓度较高 (>3 5mmol/L)时 ,以NH2 CONH2 为氮源 ,生长较快。以NH4 或NH2 CONH2 为氮源时 ,EPA(Eicosapentaenoicacid)和PUFAs占总脂肪酸的比例随着其浓度的增加而上升 ;而以NO3 -为氮源时 ,EPA和PUFAs随着NO3 -浓度增加先上升后下降 ,最适NO3 -浓度为 1 8mmol/L ,此时的EPA占总脂肪酸的比例为 16 7%。EPA占干重 (w/w)的比例 ,不管是哪种氮源 ,均随着氮浓度的增加而升高 ,但是在 0 9— 3 5mmol/L之间 ,3种氮源间EPA含量差异不显著。当氮源浓度为 7 0mmol/L时 ,以NH2 CONH2 为氮源 ,EPA和PUFAs含量最高 ,分别为 2 6 %和 4 4 %。PUFAs占干重的比例随着NO3 -浓度增加而下降 ,随NH2 CONH2 浓度增加而升高 ,而受NH4 浓度变化的影响不显著。     

EVIDENCE FOR A COMMON EVOLUTIONARY ORIGIN OF LIGHT-HARVESTING FUCOXANTHIN CHLOROPHYLL a/c-PROTEIN COMPLEXES OF PAVLOVA GYRANS (PRYMNESIOPHYCEAE) AND PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

A fucoxanthin-chlorophyll a/c-protein complex has been isolated from the prymnesiophyte Pavlova gyrans. Thylakoid membranes were treated with the mild anionic detergent sodium taurodeoxycholate followed by sucrose density gradient centrifugation. The brown fraction produced by this procedure was treated with Triton X-100 followed by a second sucrose density gradient centrifugation. A brown fraction isolated from this gradient was shown to be a light-harvesting complex nearly identical to that which is present in the diatom Phaeodactylum tricornutum. The complexes from the two organisms have nearly identical absorption and flourescence spectra, both complexes contain fucoxanthin and two other carotenoids, both contain four polypeptides of similar molecular weights, and polypeptides from both complexes cross react with antibodies raised to polypeptides of the Phaeodactylum tricornutum complex. Results suggest a common evolutionary origin for these light-harvesting complexes, in apparent contrast to the great differences in cell structure between prymnesiophytes and diatoms.     

EFFECTS OF IRON DEFICIENCY ON ISOCHRYSIS GALBANA (CHRYSOPHYCEAE) AND PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Cultures of Isochrysis galbana Parks and Phaeodactylum tricornutum Bohlin were grown in iron-limited chemostats. With increasing iron deficiency, photosynthetic rate per cell and assimilation number decreased. The pattern of photosynthesis was also altered; in Fe deficient cells the proportion of ¹⁴C fixed in glycine and serine decreased with an accompanying increase into alanine after 3 min assimilation. Although there was no significant effect of Fe deficiency on the proportion of ¹⁴C incorporated into total amino acids and amides, the percentage of total ¹⁴C fixed in protein increased with increasing Fe deficiency. Cellular levels of chlorophyll a, carotenoids, cytochromes and protein also decreased with increasing Fe deficiency. However, the reduction in chlorophyll a/cell was not as great as that of cytochrorne f₁ and Fe deficient cells therefore showed a marked increase in chlorophyll a:cytochrorne f₁ ratio.     

EFFECTS OF ENVIRONMENTAL FACTORS ON PHOTOSYNTHESIS PATTERNS IN PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE). II. EFFECT OF OXYGEN1

Oxygen inhibited the rate of light-saturated photosynthesis of the marine diatom Phaeodactylum tricornutum Bohlin. However, inhibition could only be detected with O₂ concentrations approaching 100%. Atmospheric concentrations of O₂ (21%) had little effect on photosynthesis. In this, Phaeodactylum more closely resembles the so-called C-4 plants which show low rates of photorespiration. The results presented here agree with others in showing increased O₂ inhibition at reduced bicarbonate concentrations. The biochemical mechanism of photorespiration in Phaeodactylum appears to be similar to that reported for other photosynthetic systems. The activity of ribulose-1,5 diphosphate (RuDP) carboxylase in cell-free extracts was also inhibited, by oxygen. Inhibition by O₂ was optimal at pH 9.2 as was the RuDP-dependent O₂ uptake. RuDP carboxylase/oxygenase ratios decreased with increasing pH and were greater in cells grown at lower light intensities. Carboxylase levels were less affected by the light intensity for growth than were the levels of the oxygenase. Short-term incorporation of NaHCO₃-¹⁴C by cells grown at high light intensities showed increased labelling of glycolate and glycine plus serine under O₂ compared with nitrogen. There was a concomitant decrease in the radioactivity found in phosphoglyeric acid (PGA) and sugar phosphates in the presence of O₂. The effects of O₂ on the short-term pattern of photosynthesis were less marked when the alga was previously grown at low light intensities.     

INTERACTION (ALLELOPATHY) BETWEEN MARINE DIATOMS: THALASSIOSIRA PSEUDONANA AND PHAEODACTYLUM TRICORNUTUM1

Two marine diatoms were studied singly and in mixed culture. Thalassiosira pseudonana (Hust.) Hade & Heimdal was capable of a higher growth rate (μ_max) than Phaeodactylum tricornutum Bohlin. In two-species batch cultures P. tricornutum took over in the latter portion of the exponential phase, possibly due to allelopathy. The filtrate from this species caused an initial lag phase and a reduced terminal population density for T. pseudonana. Two-species continuous cultures showed verification of these interactions. At high dilution rate (i.e., high growth rate) P. tricornutum washed out when added at low density, whereas T. pseudonana maintained constant cell density. However, when sufficient density of P. tricornutum was added as a contaminant, both species washed out. At a lower dilution rate P. tricornutum increased in density when added and eventually reached a stable population; T. pseudonana then washed out.     

INTERACTIONS OF LIGHT/DARK CYCLES AND NITROGEN PULSES ON THE TIMING OF CELL DIVISION IN THE NITROGEN-LIMITED MARINE DIATOM CYLINDROTHECA FUSIFORMIS (BACILLARIOPHYCEAE)1

Cell division in most eukaryotic algae grown on alternating periods of light and dark (LD) is synchronized or phased so that cell division occurs only during a restricted portion of the LD cycle. However, the phase angle of the cell division gate, the time of division relative to the beginning of the light period, is known to be affected by growth conditions such as nutrient status and temperature. In this study, it is shown that the phase angle of cell division in a diatom, Cylindrotheca fusiformis Reimann and Lewin, is affected by the N-limited growth rate; cell division occurred later in the dark period (12:12 h LD cycle) when the growth rate was infradian (D = 0.42 d^?1) than when it was ultradian (D = 1.0 d^?1). Nitrogen-pulses did not affect the phase angle of the division gate, but could shift the time of peak cell division activity within the division gate. The effects, if any, of N-pulses were dependent upon the growth rate and the time of day that the pulses were administered. These responses indicate that the timing of cell division in this diatom is not determined solely by the zeitgeber from the LD cycle, but rather that a LD cycle control mechanism and a N-mediated control mechanism are both involved and are somewhat interdependent. In addition, an increase in protein was observed immediately after administering a N-pulse to C. fusiformis in the ultradian growth mode indicating that the accumulation of protein can be uncoupled from the cell division cycle.     

RELATIONSHIP BETWEEN NUCLEOSIDE DIPHOSPHATE KINASE ACTIVITY AND LIGHT-LIMITED GROWTH RATE IN THE MARINE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1

Light-limited cultures of the marine diatom Thalassiosira pseudonana (Hustedt) Hasle and Heimdal (3H clone) were grown over a range of growth rates between 0.06 and 1.64 d^?1. Variations in cell volume, cell quotas of carbon, nitrogen, and protein, and maximal activity of the enzyme nucleoside diphosphate kinase (NDPK) were measured and examined as a function of growth rate. NDPK from T. pseudonana showed K_m values of 0.24 and 0.68 mM for thymidine 5′-diphosphate and adenosine 5′-triphosphate (ATP), respectively, which are similar to those found for NDPK from a variety of organisms, from bacteria to mammals. An apparent activation enthalpy of 3.52 kCal·mol^?1 was determined from Arrhenius plots. No thermodynamic transition points were noted over a temperature range from 10° to 25°C. NDPK activity was significantly correlated with growth rate but not with cell volume, carbon, nitrogen, or protein; for interspecific comparisons, normalization of enzyme activity to cell number may be most meaningful. NDPK activity per cell versus growth rate followed a U-shaped relationship, being relatively constant between 0.5 and 1.0 d^?1 and rising at higher and lower growth rates. Over this range, enzyme activity may be regulated by substrate concentration (ATP or other nucleoside triphosphates) or by adenylate energy charge. At higher growth rates where energy charge and substrate concentrations are probably high, changes in enzyme concentration appear to be required. The reasons for a rise in enzyme activity at low growth rate is unclear. Simultaneous measurement of nucleoside di- and triphosphate levels alongside NDPK measurements may help clarify the relationship, but these preliminary experiments indicate that NDPK is of limited usefulness as an index of in situ growth rate.     

GROWTH RESPONSES OF THE DIATOM,CYCLOTELLA CRYPTICA (BACILLARIOPHYCEAE), TO GIBBERELLIC ACID1

The plant hormone, gibberellic acid (GA), stimulated growth of a marine diatom, Cyclotella cryptica Reimann, Lewin and Guillard. Four concentrations of GA (5 × 20 × 25 × and 35 × 10^?6 g/mL) were added to axenic cultures of C. cryptica. Changes in cell densities, measured by cell counts and turbidimetric readings, confirmed that GA at 20 × 10^?6 g/mL produced maximum stimulation. There was an increase in the total number of cells produced and a shorter lag phase of growth at this concentration. Coulter counter measurements of cell size, as well as ocular micrometer measurements, indicated there was no significant variation in cell volumes of GA grown cells over that of the controls.     

FIRST INDUCED PLASTID GENOME MUTATIONS IN AN ALGA WITH SECONDARY PLASTIDS: psbA MUTATIONS IN THE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) REVEAL CONSEQUENCES ON THE REGULATION OF PHOTOSYNTHESIS1

Diatoms play a crucial role in the biochemistry and ecology of most aquatic ecosystems, especially because of their high photosynthetic productivity. They often have to cope with a fluctuating light climate and a punctuated exposure to excess light, which can be harmful for photosynthesis. To gain insight into the regulation of photosynthesis in diatoms, we generated and studied mutants of the diatom Phaeodactylum tricornutum Bohlin carrying functionally altered versions of the plastidic psbA gene encoding the D1 protein of the PSII reaction center (PSII RC). All analyzed mutants feature an amino acid substitution in the vicinity of the Q_B‐binding pocket of D1. We characterized the photosynthetic capacity of the mutants in comparison to wildtype cells, focusing on the way they regulate their photochemistry as a function of light intensity. The results show that the mutations resulted in constitutive changes of PSII electron transport rates. The extent of the impairment varies between mutants depending on the proximity of the mutation to the Q_B‐binding pocket and/or to the nonheme iron within the PSII RC. The effects of the mutations described here for P. tricornutum are similar to effects in cyanobacteria and green microalgae, emphasizing the conservation of the D1 protein structure among photosynthetic organisms of different evolutionary origins.     

EFFECT OF NITROGEN SOURCE ON PLATYMONAS (CHLOROPHYTA) CELL COMPOSITION AND AMINO ACID UPTAKE RATES1

The effect of nitrogen source (nitrate, ammonia and/or amino acids) on cell composition and amino acid uptake rates was examined. Substantial levels of free amino acids accumulated intracellularly with all nitrogen sources used. Ammonia accumulated only when provided in the medium. The presence of ammonia in the medium decreased the intracellular accumulation of free amino acids, especially arginine. Amino acid uptake rates were suppressed by the presence of excess nitrogen, especially ammonia. However, the suppression of uptake did not show any particular relation to the nitrogenous cell composition.     

AMINO ACID AND UREA UPTAKE IN TEN SPECIES OF CHLOROPHYTA1

The Uptake of radioartively-labelled mixed amino acids, arginine, lysine, leucine, glutamic acid and urea was examined in six species of Volvocales and four species of Chlarococcales grown in nitrate-containing medium. Nonradioactive amino acids in excess were used to estimate specificity of amino and carriers in selected cases. All ten species possess salurable (hence, carrier-mediated) systems for uptake of both arginine and urea. In all Volvacales and one Chlorococcales, the arginine-speciftc carrier (which also transported lysine with lower efficiency) was the only amino acid carrier detected. Three species of Chlororoccales appear to possess a separate carrier for lysine and two of these appear to possess at least one additional carrier that is involved in uptake of non-basic amino acids.     

EFFECTS OF SILICON DEFICIENCY ON LIPID COMPOSITION AND METABOLISM IN THE DIATOM CYCLOTELLA CRYPTICA1

The effects of silicon deficiency on the metabolism and composition of lipids in Cyclotella cryptica T13L Reimann, Lewin, and Guillard were examined. Silicon-deficient cells had higher levels of neutral lipids (primarily triacylglycerols) and higher proportions of saturated and monounsaturated fatty acids than silicon-replete cells. After 4 h of silicon deficiency, the percentage of newly assimilated NaH¹⁴CO₃ partitioned into lipids increased from 27.6% to 54.1%, whereas the percentage partitioned into chrysolaminarin decreased from 21.6% to 10.6%. In addition, pulse-chase experiments with NaH¹⁴CO₃ indicated that the amount of ¹⁴C in the total cellular lipid fraction increased by 32% after 12 h of silicon deficiency despite the absence of additional photoassimilable ¹⁴C. Therefore, the accumulation of lipids in response to silicon deficiency appears to be due to two distinct processes: (a) an increase in the proportion of newly assimilated carbon partioned into lipids, and (2) a slow conversion of previously assimilated carbon from non-lipid compounds into lipids