Biosynthesis of Phytochelatins and Arsenic Accumulation in the Marine Microalga <Emphasis Type="BoldItalic">Phaeodactylum tricornutum</Emphasis> in Response to Arsenate Exposure

The arsenate-induced synthesis of phytochelatins (PC), intracellular cysteine-rich metal-binding peptides, and its relationship with toxicity and with As accumulation in the cell have been studied in laboratory cultures of the marine microalga Phaeodactylum tricornutum. The time course of cellular PC and As in short-term exposures showed that the involvement of PC in the As detoxification as well as the pathway of cellular As depend on the extent of As accumulation and on the rate of PC synthesis. At arsenate concentrations causing As accumulation at a rate exceeding that of PC synthesis, cells seem to activate a mechanism of release of As mainly in a chemical form not complexed with PC. At arsenate concentrations at which the synthesis of PC occurs at a rate sufficient to allow a significant portion of As accumulated in the cell to be bound, the fate of cellular As seems to be mainly controlled by PC. The occurrence of these different pathways of As detoxification was discussed to explain the pattern of cellular As and PC in cells grown for three days at growth-inhibitory and at no growth-inhibitory concentration of arsenate.     

NONDESTRUCTIVE TRACING OF MIGRATORY RHYTHMS OF INTERTIDAL BENTHIC MICROALGAE USING IN VIVO CHLOROPHYLL A FLUORESCENCE1,2

In vivo chlorophyll (Chl) a fluorescence was measured in undisturbed intertidal sediments with the purpose of tracing the vertical migratory rhythms of benthic microalgae. A pulse amplitude fluorometer, an instrument which does not require physical contact with the sample, was used, thus allowing successive measurements to be taken on the same sample without causing any type of disturbance to the sediment structure. The basis of the method is the possibility to detect changes in the Chl a concentration near the sediment surface caused by the vertical movement of the microalgae. This requires the verification of two conditions: the possibility to follow changes in the sediment Chl a content from fluorescence intensity, and a sediment photic depth smaller than the vertical distances covered by the moving microalgae. Both conditions were experimentally verified in intertidal muddy sediments of the Tagus estuary, Portugal. In vivo fluorescence was shown to vary linearly with the sediment Chl a content, and the sediment photic depth was estimated to reach 0.27 mm, a value clearly smaller than the reported depths for microalgal migrations. Sediment samples kept under in situ conditions exhibited large hourly Variations (over 400%) in the Chl a fluorescence intensity, which were closely synchronized with the daytime periods of emersion. The rhythmic fluctuations in Chl a fluorescence were confirmed further to represent microalgal migration by (1) its endogenous nature (fluorescence continued to follow diurnal and tidal cycles after removal of environmental stimuli), (2) its dependence on the vertical distribution of the microalgal population within the sediment (vertically homogenized samples failed to display fluorescence variations), and (3) the lack of significant temperature and light effects on the fluorescence emission under in situ conditions (tested in three species representative of the main groups found in the studied microphytobenthic communities—the diatom Phaeodactylum tricornutum (Böhlin), the cyanobacterium Spirulina maxima (Setch. et Gard.), and the euglenophyte Euglena granulata (Klebs) Lemm.). The results obtained indicate that, in spite of the potential concurrent effects of factors other than the Chl a concentration on the fluorescence intensity, in vivo Chl a fluorescence can be used to trace nondestructively the migratory behavior of benthic microalgae.     

SILICON UPTAKE BY ALGAE WITH NO KNOWN Si REQUIREMENT. II. STRONG pH DEPENDENCE OF UPTAKE KINETIC PARAMETERS IN PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Silicon uptake kinetics of the diatom Phaeodactylum tricornutum (Bohlin) were examined at pH 8.8 ± 0.1 and pH 9.1 ± 0.1. Uptake follows hyperbolic saturation kinetics at both pH's, but at the higher pH the half-saturation constant for uptake is 11.8 μM, as opposed to 54.8 μM at the lower pH. When the uptake rate is examined as a function of the calculated concentration of the monovalent conjugate base, SiO(OH)₃^?, the half-saturation constant for uptake is 6.6 μM at either pH.     

LIGHT DEPENDENCE OF GROWTH AND PHOTOSYNTHESIS IN PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Phaeodactylum tricornutum Bohlin was maintained in exponential growth over a range of photon flux densities (PFD) from 7 to 230 μmol·m^?2s^?1. The chlorophyll a-specific light absorption coefficient, maximum quantum yield of photosynthesis, and C:N atom ratio were all independent of the PFD to which cells were acclimated. Carbon- and cell-specific, light-satuated, gross photosynthesis rates and dark respiration rates were largely independent of acclimation PFD. Decreases in the chlorophyll a-specific, gross photosynthesis rate and the carbon: chlorophyll ratio and increases of cell- or carbon-specific absorption coefficients were associated with an increase in cell chlorophyll a in cultures acclimated to low PFDs. The compensation PFD for growth was calculated to be 0.5 μmol·m^?2s^?1. The maintenance metabolic rate (2 × 10^?7s^?1), calculated on the basis of the compensation PFD, is an order of magnitude lower than the measured dark respiration rate(2.7 × 10^?6mol O₂·mol C^?1s^?1). Maintenance of high carbon-specific, light-saturated photosynthesis rates in cells acclimated to low PFDs may allow effective use of short exposures to high PFDs in a temporally variable light environment.     

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.     

CELL DIVISION PERIODICITY IN 13 SPECIES OF MARINE PHYTOPLANKTON ON A LIGHT: DARK CYCLE1

The division rates of 26 clonal cultures representing 13 species of planktonic marine algae (6 diatoms, 2 flagellated chrysophytes, 2 coccolithophores, 1 cryptomonad flagellate, I dinoflagellate, 1 green alga) were determined every 2 h for 48 h during exponential growth on a 14:10 LD cycle in nutrient-replete batch culture. Cyclic oscillations in the division rate were detectable in 22 of these clones. Of 14 diatom clones examined, four displayed nearly constant division rates throughout the LD cycle and ten showed strong periodicity favoring division during the light periods. In contrast, all other algae (12 clones) exhibited division rate maxima during periods of darkness, and clearly detectable decreases in cell number for time intervals of 4–8 h during periods of illumination. Intraspecific differences in division periodicity were found among eight clones of the diatom Thalassiosira pseudonana (Hustedt) Hasle & Heimdal and six clones of the coccolithophore Emiliania huxleyi (Lohm.) Hay & Mohler.     

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.     

Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes

A general purpose transformation vector, designated pPha-T1, was constructed for use with the diatom Phaeodactylum tricornutum Bohlin. This vector harbors the sh ble cassette for primary selection on medium containing the antibiotic zeocin, and a multiple cloning site flanked by the P. tricornutum fcp A promoter. pPha-T1 was used to establish the utility of three selectable marker genes and two reporter genes for P. tricornutum transformation. The nat and sat-1 genes confer resistance to the antibiotic nourseothricin, and npt II confers resistance to G418. Each of these genes was effective as a selectable marker for identifying primary transformants. These markers could also be used for dual selections in combination with the sh ble gene. The reporter genes uid A and gfp were also introduced into P. tricornutum using pPha-T1. Gus expression in some transformants reached 15 μg·μg⁻¹ of total soluble protein and permitted excellent cell staining, while GFP fluorescence was readily visible with standard fluorescence microscopy. The egfp gene, which has optimal codon usage for expression in human cells, was the only version of gfp that produced a strong fluorescent signal in P. tricornutum. The codon bias of the egfp gene is similar to that of P. tricornutum genes. This study suggests that codon usage has a significant effect on the efficient expression of reporter genes in P. tricornutum. The results presented here demonstrate that a variety of selectable markers and reporter genes can be expressed in P. tricornutum , enhancing the potential of this organism for exploring basic biological questions and industrial applications.     

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.     

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.