NITRATE REDUCTASE ACTIVITY OF AMPHIDINIUM CARTERI AND CACHONINA NIEI (DINOPHYCEAE) IN BATCH CULTURE: DIEL PERIODICITY AND EFFECTS OF LIGHT INTENSITY AND AMMONIA1,2

The activity of extracted NADH-NO₃^? reductase was measured in the marine dinoflagellates Amphidinium carteri Hulburt and Cachonina niei Loeblich. Its activity showed a diel periodicity and was ca. twice as great at midday as at midnight. The enzyme activity was unstable, with an in vitro half-life of 2–3 h. Values of enzyme activity were low or undetectable during lag phase but paralleled the instantaneous growth rate value during log phase. Nitrate reductase activity was not found in the stationary phase of growth, but additions of NO₃^? resulted in enzyme activity after 24h. When A. carteri was exposed to a series of light intensities for several weeks, the division rate and enzyme activity increased with increasing light intensity up to saturating intensities. In 6 h exposures, enzyme activity decreased with decreasing light intensities below light intensities saturating division rate. Additions of NH₄⁺ (0.5–50 μm) to A. carteri cultures decreased the amount of extractable enzyme. The in vitro activity was not inhibited by similar NH⁺₄ concentrations.     

PHOSPHATE ACCUMULATION AND METABOLISM BY HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE) DURING DIEL VERTICAL MIGRATION IN A STRATIFIED MICROCOSM1

Diel vertical migration by Heterosigma akashiwo (Hada) Hada (Raphidophyceae) was monitored in a 1.5 in tall microcosm. Vertical stratification, with low salinity and low orthophosphate (P_i) concentration in the upper layer and high salinity and high P_i concentration in the lower layer, was simulated in the tank, analogous to summer stratification in the Seto Inland Sea. The phosphate metabolism of H. akashiwo during this vertical migration was studied using ³¹P-NMR spectroscopy. At night this species migrated to the lower phosphate-rich layer and took up inorganic phosphate (P_i) which then was accumulated as polyphosphate (PP_i) by an increase in the chain length of PP_i During the daytime this species migrated to the phosphate-depleted surface water and utilized the accumulated PP_i for photophosphorylation by decreasing the chain length of PP_i During the first night after the phosphorus was introduced to the previously impoverished waters, the cells took up inorganic phosphate, accumulating the new phosphorus nutrient internally as P_i But the cells did not convert P_i to PP_i presumably due to their lack of ATP. After the second day of the experiment, conversion of P_i to PP_i at night was much more rapid than on the first day, presumably due to increased ATP availability. Then the cycle continued, with uptake of P_i and conversion to PP_i at night at the bottom and its utilization during the day at the surface. These data suggest that the role of PP_i in the metabolism of this species appears to be as a phosphate pool which regulates the level of P_i and ATP in the cell. Diel vertical migration allows this red tide species to shuttle between the phosphate-rich lower layer and the photic upper layer in stratified waters. ³¹P-NMR is shown to be a valuable tool in studying the phosphorus metabolism in migrating organisms.     

DIEL CHANGES IN PHASED-DIVIDING CULTURES OF CERATIUM FURCA (DINOPHYCEAE): NUCLEOTIDE TRIPHOSPHATES,ADENYLATE ENERGY CHARGE,CELL CARBON,AND PATTERNS OF VERTICAL MIGRATION1

The diel pattern of cell division, cell carbon, adenine nucleotides and vertical migration was determined for laboratory cultures of the photosynthetic marine dinoflagellate, Ceratium furca (Ehr.) Clap. & Lachm., entrained on an alternating 12:12 LD schedule at 20 C. Cell division was initiated during the latter portion of the dark period with ca. 30% of the population undergoing division. Cell C increased during the light period and exhibited a linear decrease with a loss of 33% during the dark period. ATP · cell^?1 increased during the light period and decreased by ca. 40–50% during the dark period. The diel patterns of cell C and ATP tended to “buffer” the magnitude of the change in C:ATP ratios around an overall mean value of 89. There was no obvious trend in the concentration of [GTP + UTP] · cell^?1 over the cell cycle. The cellular adenylate energy charge was maintained at values between 0.8 to 0.9 throughout the 24 h LD cycle, despite a ca. 40% decrease in total adenylates (A_T= ATP + ADP + AMP) during the dark period on 12:12 LD, and over a 68% decrease in ATP during 42 h of continuous darkness. These data lend experimental support to the theory of cellular metabolic control by the adenine nucleotides. With lateral illumination on 12:12 LD cycles, the cells began to concentrate at the surface of the experimental tubes shortly before the lights were turned on, and at the bottom of the tubes shortly before the lights were extinguished. This pattern continued for 6 days in continuous darkness, suggesting that the vertical migration pattern is independent of a phototactic response and may be under the control of an endogenous rhythm.     

THE RESPONSE OF GROWTH AND BIOCHEMICAL COMPOSITION TO VARIATIONS IN DAYLENGTH, TEMPERATURE, AND IRRADIANCE IN THE MARINE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)

The relationships between the growth rate of the marine diatom Thalassiosira pseudonana (Hustedt) and irradiance, daylength, and temperature were determined in nutrient-sufficient semicontinuous cultures. The initial slopes of the growth versus total daily irradiance curves were not affected by temperature or daylength. Growth versus irradiance was best modeled as a hyperbolic function at short daylengths and better modeled as an exponential function at longer daylengths. The maximum or light-saturated growth rates at each daylength were modeled as a hyperbolic function of daylength. This model was extended in a novel manner to include temperature dependence providing a framework that can be used to interpret other experimental data on growth rate versus daylength. The resulting model should be useful in global models of phytoplankton growth. Carbon, nitrogen, and chl a quotas were influenced by daylength, irradiance, and temperature. Both C and N quotas were positive exponential functions of irradiance, whereas N and chl a quotas were significantly greater for cells grown at the lower temperature. The ratio chl a :C quota (chl a :Q_c) was a strong negative exponential function of total daily irradiance. Cells grown at 10° C had significantly greater chl a :Q_c ratios than those grown at 18° C, and daylength also had a significant positive influence on chl a :Q_c. The apparent effect of daylength on chl a :Q_c was removed by standardizing chl a :Q_c to growth rate (μ), resulting in a temperature-dependent relationship between chl a :Q_c·μ⁻¹ and irradiance that accounted for 95% of the variation in the data.     

CHEMICAL COMPOSITION AND PHOTOSYNTHETIC CHARACTERISTICS OF ETHMODISCUS REX (BACILLARIOPHYCEAE): EVIDENCE FOR VERTICAL MIGRATION1

Ethmodiscus rex (Rattray) Wiseman and Hendey cells from near surface net tows in the Southwest Atlantic Ocean and Caribbean Sea were examined for chemical composition, internal nutrient pool concentrations, and oxygen evolution characteristics. Elemental ratios indicated nitrogen limitation with C:N:P ratios of 125:9:1 (atoms), and carbon: chlorophyll (chl) ratios of 129:1 (weight). However, internal nitrate pools (1.4–27.1 mM) suggested that cells were not N-limited. Intracellular NO₃^? accounted for up to 54% (range = 3–54%) of the total N quota in some samples. Photosynthetic parameters were consistent with a high-light-adapted population and suggested an instantaneous maximum chl-specific photosynthetic rate (P^B_max) of 4.8–12.4 nmol O₂·μg chl^?1· h^?1. Respiration rates varied ten-fold and were inversely related to P^B_max Ethmodiscus chemical composition and buoyancy characteristics are similar to vertically migrating Rhizosolenia mats and the non-motile dinoflagellate Pyrocystis noctiluca Murray (Schuett). The presence of internal NO₃^? pools in Ethmodiscus suggests that this genus is also vertically migrating to exploit sub-surface nitrogen pools. Such behavior may be widespread in large, non-motile oceanic phytoplankton. Based on ascent rate data, chemical composition, and photosynthetic rates, we estimate that the entire division–migration cycle for Ethmodiscus requires at least 7–12 days.     

CA2+ BINDING PROTEIN CALRETICULIN IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA): BIOCHEMICAL CHARACTERIZATION, DIFFERENTIAL EXPRESSION DURING SEXUAL REPRODUCTION, AND PHYLOGENETIC ANALYSIS

The occurrence of calreticulin, the main Ca²⁺ binding protein in the endoplasmic reticulum of eukaryotic cells, was investigated in the unicellular green alga Chlamydomonas reinhardtii Dangeard. The biochemical characterization of a diethylaminoethyl purified extract highlighted the presence, on SDS-PAGE, of a 55-kDa protein that stained blue with the Stains All dye, a diagnostic feature of acidic Ca²⁺ binding proteins. Immunoblot analyses revealed a strong cross-reaction of the Chlamydomonas reinhardtii protein with antibodies to plant calreticulins and the endoplasmic reticulum retention signal HDEL. Furthermore, the 55-kDa protein bound [⁴⁵Ca²⁺] and had an acidic isoelectric point (pI = 4.9) but was neither glycosylated nor phosphorylated. N-terminal sequencing revealed strong amino acid sequence similarity to calreticulin from other sources. The presence of calreticulin in Chlamydomonas reinhardtii suggested that an endoplasmic reticulum Ca²⁺ buffering mechanism was present in this unicellular chlorophyte. The data suggest an early origin and high conservation of endoplasmic-reticulum-mediated Ca²⁺ functions in eukaryotes, whereby specific posttranslational modifications of the proteinhave been specifically acquired in different lineages of photosynthetic eukaryotes. Moreover, northern and western blot analysis experiments showed a regulation of calreticulin expression during Chlamydomonas sexual reproduction with a high abundance of calreticulin mRNA and protein in reproductive cells.