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.     

PIGMENT TURNOVER IN THE MARINE DIATOM THALASSIOSIRA WEISSFLOGII. I. THE 14CO2-LABELING KINETICS OF CHLOROPHYLL a1

The turnover of chlorophyll a (chl a) was investigated in the diatom Thalassiosira weissflogii (Grunow) Fryxell and Hasle using a new method based on the incorporation of ¹⁴C into chl a. The alga was maintained in its exponential growth phase under continuous light; ¹⁴C was supplied as bicarbonate. The time course of label accumulation into the tetrapyrrole ring and the phytol side chain was determined for time periods equivalent to 1–2 cell doublings. The labeling kinetics of the tetrapyrrole ring and the phytol side chain were described satisfactorily by a simple precursor-pigment model with two free parameters, the precursor turnover rate and the pigment turnover rate, both having dimensions of time^?1. The model was fit to the experimental data to determine the values of these two free parameters. The turnover rates of the tetrapyrrole ring and the phytol side chain were not significantly different, ranging from 0.01 to 0.1 per day. These rates are equivalent to turnover times ranging from days to weeks. Growth rate-normalized turnover rates did not vary with irradiance (7.5–825 μE · m^?2· s^?1). The precursor turnover rates of the tetrapyrrole ring and the phytol side chain differed by an order of magnitude. These results indicate that chl a is not degraded significantly in cultures of T. weissflogii grown under continuous light. Neither irradiance nor growth rate affected growth rate-normalized chlorophyll turnover rates. Our results are inconsistent with the hypothesis that steady-state cellular concentrations of chl a are maintained by a dynamic equilibrium between rates of synthesis and degradation.     

STRUCTURAL FEATURES OF NUCLEAR GENES IN THE CENTRIC DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)

Thalassiosira weissflogii (Grun.) Fryxell et Hasle is one of the more commonly studied centric diatoms, and yet molecular studies of this organism are still in their infancy. The ability to identify open reading frames and thus distinguish between introns and exons, coding and noncoding sequence is essential to move from nuclear DNA sequences to predicted amino acid sequences. To facilitate the identification of open reading frames in T. weissflogii , two newly identified nuclear genes encoding β-tubulin and t  -complex polypeptide (TCP)-γ, along with six previously published nuclear DNA sequences, were examined for general structural features. The coding region of the nuclear open reading frames had a G + C content of about 49% and could readily be distinguished from noncoding sequence due to a significant difference in G + C content. The introns were uniformly small, about 100 base pairs in size. Furthermore, the 5' and 3' splice sites of introns displayed the canonical GT/AG sequence, further facilitating recognition of noncoding regions. Six of the nuclear open reading frames displayed relatively little bias in the use of synonymous codons, as exemplified by the cDNAs encoding β-tubulin and TCP-γ. Two open reading frames displayed strong bias in the use of particular codons (although the codons used were different), as exemplified by the cDNA encoding fucoxanthin chlorophyll a/c binding protein. Knowledge of codon bias should facilitate, for example, design of degenerate PCR primers and potential heterologous reporter gene constructs.     

PIGMENT TURNOVER IN THE MARINE DIATOM THALASSIOSIRA WEISSFLOGII. II. THE 14CO2-LABELING KINETICS OF CAROTENOIDS1

The biosynthesis and turnover of the pigments fucoxanthin, diadinoxanthin (DD), and diatoxanthin (DT) were studied in exponentially growing cultures of the diatom Thalassiosira weissflogii (Grunow) Fryxell and Hasle to investigate the dependence of pigment turnover on algal growth rates and light intensity. ¹⁴C-bicarbonate was used as a tracer. The labeling kinetics of fucoxanthin and DT were described satisfactorily by a simple precursor-pigment model with two free parameters, the precursor and pigment turnover rate. At growth irradiances < 200 μE · m^?2· s^?1, labeling kinetics of DD indicated the presence of two kinetically distinct DD pools and at least one precursor pool. The average growth rate-normalized pigment turnover rate of fucoxanthin was 0. The growth rate-normalized turnover rate of DT, determined only at high light irradiances (> 200 μE·m^?2·s^?1), was 1.3. At high light irradiances, the growth rate-normalized turnover rate of DD was 1.8. At low light irradiances, the turnover rates of the two DD pools were 3.7 and 0, respectively. The corresponding pigment turnover times were on the order of days to weeks, depending on the growth rate of the cultures. A comparison of pigment pool sizes, pigment turnover rates, and precursor turnover rates suggests that fucoxanthin is synthesized from a pool of DD and that DD and DT are synthesized from a common precursor, possibly β-carotene. No evidence was seen for dynamic xanthophyll cycling. This suggests that the commonly known “xanthophyll cycle” is the simple unidirectional conversion of DD into DT, or of DT into DD, in response to rapid irradiance changes.     

FLOW CYTOMETRIC ANALYSIS OF SPERMATOGENESIS IN THE DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)1

Flow cytometry was used to detect and quantify sexual differentiation in the centric diatom Thalassiosira weissflogii (Grun.). Size (light scatter), chlorophyll, protein and DNA contents were measured for each cell throughout the process of differentiation. Male gametes were small round cells characterized by one complement of DNA and a lower protein and chlorophyll content than vegetative cells. Male gamete formation was induced by a long period of darkness (2 days) followed by a transfer to continuous light. Up to 30% of the initial cell population produced male gametes which appeared in the culture 14 h after release from darkness. Male gamete production was also detected in exponentially growing cultures in continuous light, but to a much smaller degree.     

FLOW CYTOMETRIC ANALYSIS OF SPERMATOGENESIS IN THE DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)1

Flow cytometry was used to detect and quantify sexual differentiation in the centric diatom Thalassiosira weissflogii (Grun.). Size (light scatter), chlorophyll, protein and DNA contents were measured for each cell throughout the process of differentiation. Male gametes were small round cells characterized by one complement of DNA and a lower protein and chlorophyll content than vegetative cells. Male gamete formation was induced by a long period of darkness (2 days) followed by a transfer to continuous light. Up to 30% of the initial cell population produced male gametes which appeared in the culture 14 h after release from darkness. Male gamete production was also detected in exponentially growing cultures in continuous light, but to a much smaller degree.     

SILICON DEPOSITION DURING THE CELL CYCLE OF THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE) DETERMINED USING DUAL RHODAMINE 123 AND PROPIDIUM IODIDE STAINING1

The relatively non-toxic dye, rhodamine 123 (R123), was incorporated into the frustule of Thalassiosira weissflogii Grun. clone ACTIN in direct proportion to biogenic silica (BSi). R123 was used together with the DNA stain propidium iodide to track and quantify Si deposition during the cell cycle of T. weissflogii using flow cytometry. Silicon deposition was not continuous through the cell cycle. Deposition of the valves occurred during M phase. The hypocingulum was largely deposited during G1 with some suggestion of minor girdle band deposition during G2. Silicon deposition did not occur during S phase. Assuming that a complete frustule consists of an epivalve, epicingulum, hypocingulum, and hypovalve, then 40% of cellular BSi was contained within the cingulum of T. weissflogii with 60% present in the valves. These percentages correspond to 0.38 pmol Si in the two cingula and 0.57 pmol Si in the valves. Temporal differences in the timing of silicic acid uptake and deposition during the cell cycle of T. weissflogii suggested that deposition of both the new valves and the cingulum is supported by an internal pool of dissolved Si acquired during G2.     

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 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.     

NTAGONISM BETWEEN CADMIUM AND IRON IN THE MARINE DIATOM THALASSIOSIRA WEISSFLOGII1

Cadmium inhibits iron uptake and assimilation in the coastal diatom Thalassiosira weissflogii Grun. The effect of cadmium on short term Fe uptake fits ft competitive binding model: where (Fe³+) and (Cd²++) tire the free ferric and cadmium ion concentrations, respectively. The apparent binding constant K_cds, is calculated to be ca. lO^8.2M^-1 compared to a K^fe of lO¹⁹ M^-1. At low free ferric ion concentrations. interference of cadmium with iron transport (at pCd = 8 and pFe* < 20) results in a simultaneous decrease in growth rate and Fe accumulation to a level known 1o limit growth. Upon decreasing the free cadmium ion concentration, cells accumulate a large amount oj iron prior to resumption of normal growth. At higher free ferric ion concentrations (pFe* < 20) normal or elevated Fe quotas are absented but “luxury consumption” of iron still occurs upon reversal of toxicity. Evidence that these algae with high cellular iron quotas are effectively Fe deficient is provided by a decrease in the cytochrome f/chlorophyll a ratio and a much greater decrease in NO₃- reductase activity than in aldolase activity or H¹⁴C0₃ assimilation. Under the conditions of this study, cadmium had little effect on Si accumulation. The transport of methylamine (an analog of NH⁺₄) is unaffected by short term exposure to high free cadmium ion concentration but is greatly inhibited upon long term (97 h) exposure.     

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.     

EFFECT OF SILICATE LIMITATION ON VALVE MORPHOLOGY IN THALASSIOSIRA AND COSCINODISCUS (BACILLARIOPHYCEAE)1

Coscinodiscus radiatus Ehrenb. and Thalassiosira eccentrica (Ehrenb.) Cleve were grown in a silicate-limited chemostat at silicate concentrations below 1 μg-atoms · l^?1. The resulting abnormal valves of C. radiatus lacked a thickened ring around the foramina; their pore membranes were thinner and their loculi shallower than those in normal cells. Abnormal valves of T. eccentrica had a fasciculate areolae pattern; they lacked a silica covering over the foramina and some tangential areolae walls. Neither abnormal valve could be termed a new species.     

CARBONIC ANHYDRASE IN THE CHLOROPLAST OF A COCCOLITHOPHORID (PRYMNESIOPHYCEAE)1

The activity and subcellular distribution of carbonic anhydrase in a coccolithophorid alga, CCMP 299, was examined. The enzyme could not be detected in crude cell homogenates but was present at high specific activity (27.5 unit·mg^?1 protein) in chloroplasts (density, 1.14 g·cm^?3) isolated in a sucrose gradient. The carbonic anhydrase activity was sensitive to known inhibitors. Inhibition at 50% (I₅₀) was obtained with concentrations of 4.60 mM and 2.65 mM for acetazolamide and NaN₃, respectively. These levels are more consistent with patterns of inhibition previously observed for chloroplastic (as compared to periplasmic) carbonic anhydrase. In this organism, carbonic anhydrase was localized in the chloroplast stroma. These findings are discussed in terms of the relationship among dissolved inorganic carbon interconversions, photosynthesis, and calcification.     

THALASSIOSIRA ANDAMANICA SP. NOV. (BACILLARIOPHYCEAE), A NEW DIATOM FROM THE ANDAMAN SEA (THAILAND)

A new marine diatom, Thalassiosira andamanica, is described from light and electron microscopy. The specimens were collected in the vicinity of Phuket Marine Biological Center, Thailand, and later brought into clonal culture. Thalassiosira andamanica possess a rimoportula with a pronounced outer extension, one marginal ring of fultoportulae, and three rings of fultoportulae on the valve face. Cells are united into colonies by a single thread secreted through a central fultoportula. Marginal fultoportulae extensions are shortest on the inside of the valve. The areolae are arranged in sectors, and the valve margin is ribbed with approximately 38 ribs in 10 μm. The valvocopula and copula have rows of pores, four to six pores in 1 μm. Apparently, the pleurae are hyaline. Experiments with a clonal culture isolated at Phuket, Thailand, showed that growth (cell divisions·24 h⁻¹) was reduced for cultures grown at 14° C compared to those grown at 19°, 24°, or 30° C. The maximum growth rate (2.2 divisions·24 h⁻¹) was at 30° C. Thalassiosira andamanica is compared with morphologically similar taxa. On the basis of morphological features and the response to different temperature regimens, it is concluded that this taxon must be recognized as a new warm-water species. In addition, T. andamanica does not clearly belong to any of the two subgroups of species of Thalassiosira. To accommodate the morphological characteristics of T. andamanica, the establishment of a possible third subgroup is discussed.     

THE RELATIONSHIP BETWEEN VALVE DIAMETER AND NUMBER OF CENTRAL FULTOPORTULAE IN THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)1

Cells well below the reported minimum size for Thalassiosira weisslogii (Grunow) Fryxell et Hasle were observed in two unialgal cultures. The relationship between diameter and number of central fultoportulae was found to be very regular. Regresion analysis of three different populations showed equivalent slopes and r² values above 80%. The y-intercepts were significantly different and support observations in the litrature that nonmarine valeve of T. Weissflogii have fewer central fultoportulae than marine forms.     

THE EFFECT OF SODIUM CHLORIDE ON CARBONIC ANHYDRASE ACTIVITY IN MARINE MICROALGAE1

The effects of NaCl on the internal and external carbonic anhydrase (CA) activity of several marine microalgae were studied. Unlike freshwater microalgae in which CA activity is generally inhibited by NaCl, marine microalgae exhibited considerable species-dependent variation when exposed to NaCl. CA activity in Phaeodactylum tricornutum, a diatom, was inhibited, whereas it was activated in the coccolithophorid Pleurochrysis carterae. CA activity in the chlorophyte Dunaliella primolecta was not significantly affected by NaCl. In Dunaliella salina and Dunaliella parva, NaCl inhibited external CA without affecting the internal activity, whereas in Chlorella vulgaris C-133 and Dunaliella peircei only the internal CA was inhibited. Internal CA of Dunaliella tertiolecta was not affected by NaCl, but the external enzyme was significantly enhanced. Salt substitution experiments revealed that chloride (Cl^-) is the ion affecting CA activity; the effect of Cl^- can be replaced by bromide ion. Cl^- affects external CA activity while also affecting the apparent affinity for inorganic carbon during photosynthesis. Microalgae whose internal CA activity was enhanced by Cl^- showed higher intracellular Cl^- concentrations than those species that were inhibited.     

AN ECOLOGICAL BASIS FOR EXTRACELLULAR CARBONIC ANHYDRASE IN MARINE UNICELLULAR ALGAE

Extracellular carbonic anhydrase (CA_e) is expressed by many, but not all, autotrophic species of aquatic unicellular protists. We measured CA_e activities in unicellular marine algae characteristic of either high nutrient spring, fall, and winter blooms or low nutrient summer populations to provide ecological/evolutionary information about the enzyme. Highest activities occurred in spring bloom and opportunistic diatoms exposed to long photoperiods (16 h) when pH was highest and CO₂ was lowest. Lower activities were recorded for a fall-bloom diatom exposed to the long photoperiod, and lowest values were found under all culture conditions for one diatom and a number of flagellated species typical of summer low nutrient environments. Other potential sources of variance in measurements of CA_e activity were examined. Maximum activities of CA_e were recorded for the diatom, Skeletonema costatum (Greville) Cleve, during late exponential phase of growth and within 8 h of the beginning of the photoperiod. We concluded that ecological factors are important in determining CA_e activities in marine unicellular protists. Potential functions of CA_e in the metabolism of marine unicellular algae are discussed.     

EFFECT OF CARBONIC ANHYDRASE INHIBITORS ON THE INORGANIC CARBON UPTAKE BY PHYTOPLANKTON NATURAL ASSEMBLAGES1

The role of carbonic anhydrase (CA) in inorganic carbon acquisition (dissolved inorganic carbon, DIC) was examined in Alboran Sea phytoplankton assemblages. The study area was characterized by a relatively high variability in nutrient concentration and in abundance and taxonomic composition of phytoplankton. Therefore, the relationship between environmental variability and capacity for using HCO₃^? via external CA (eCA) was examined. Acetazolamide (AZ, an inhibitor of eCA) inhibited the primary productivity (PP) in 50% of the samples, with inhibition percentages ranging from 13% to 60%. The AZ effect was more prominent in the samples that exhibited PP >1 mg C · m^?3 · h^?1, indicating that the contribution of eCA to the DIC photosynthetic flux was irrelevant at low PP. The inhibition of primary productivity by AZ was significantly correlated to the abundance of diatoms. However, there was no a relationship between AZ effect and CO₂ partial pressure (pCO₂) or nutrient concentration, indicating that the variability in the PP percentage supported by eCA was mainly due to differences in taxonomic composition of the phytoplankton assemblages. Ethoxyzolamide (EZ, an inhibitor of both external and internal CA) affected 13 of 14 analyzed samples, with PP inhibition percentages varying from 50% to 95%. The effects of AZ and EZ were partially reversed by doubling DIC concentration. These results imply that CA activity (external and/or internal) was involved in inorganic carbon acquisition in most the samples. However, EZ effect was not correlated with pCO₂ or taxonomic composition of the phytoplankton.