GROWTH CHARACTERISTICS OF PHYTOPLANKTON DETERMINED BY CELL CYCLE PROTEINS: PCNA IMMUNOSTAINING OF DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

By immunohistochemistry and immunofluorescence methods, we observed that the analog of proliferating cell nuclear antigen (PCNA) in Dunaliella tertiolecta Butcher (Chlorophyceae) was exclusively located in the nucleus. Among positively stained cells, PCNA abundance varied, being highest in S-phase cells, lower in others, and undetectable in early G1- or late M-phase cells. In exponentially growing and partially synchronized cultures, the percentage of PCNA-stained cells (% PCNA-stained cells) oscillated in the photocycle (12:12 h LD). It increased during the light period and reached a peak (75%) before the onset of the dark period when the culture was mainly (71%) in the S phase of the cell cycle. The DNA synthesis inhibitor, hydroxyurea, depressed PCNA abundance, whereas no effect was detected for the mitosis inhibitor colchicine. We conclude that PCNA in D. tertiolecta is associated with the S phase of the cell cycle where it is accumulated and functioning. PCNA was used to characterize the growth pattern of cultures grown in different media, temperatures, and growth stages. The time lag between the PCNA-stained phase and the M phase was very short in a continuous culture grown in reduced f/2 medium at 22°C and was considerably longer in the cultures grown in f/2 at 15°C. When an exponentially growing culture grew older, % PCNA-stained cells decreased. In a late stationary culture where there was no net growth, a small number of cells were still cycling through the PCNA-stained phase and cell division. In the continuous culture grown at 22°C, the duration of the PCNA-stained phase (T_s) was 13 h. Calculations with this T_s and % PCNA-stained cells yielded a growth rate of 0.77 d^?1, which was close to that obtained by cell counts (0.69 d^?1). Taken together, the results suggest that PCNA is a useful indicator of growth status and a promising cell cycle marker for estimation of species-specific growth rate.     

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.     

DETECTION OF PROLIFERATING CELL NUCLEAR ANTIGEN ANALOG IN FOUR SPECIES OF MARINE PHYTOPLANKTON1

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein for polymerase-δ and therefore is essential for cellular DNA synthesis. The synthesis and abundance of PCNA in the cell are cell-cycle-dependent, both increasing markedly during the S phase. Such a protein could be a useful cell cycle marker, which is required for estimating algal species-specific growth rates via the cell cycle approach. By using commercially available monoclonal anti-rat-PCNA antibody and an enhanced chemiluminescence technique, PCNA-like proteins were detected in four species of marine phytoplankton. The strong single band detected on western blots of Isochrysis galbana Parke, Thalassiosira weissflogii Cleve, and Dunaliella tertiolecta Butcher had an apparent molecular weight of 33–36 kDa. This molecular weight is within the range as observed for PCNA in a wide phylogenetic array of organisms (33–36 kDa). In the diatom Skeletonema costatum (Grev.) Cleve, the PCNA antibody detected a major band of about 19 kDa as well as a minor band of 38 kDa. The detected proteins were specifically recognized by the monoclonal anti-rat-PCNA antibody. The PCNA-like proteins in I. galbana, T. weissflogii, and D. tertiolecta were more abundant in the exponential growth stage and then decreased and became undetectable in the late stationary stage. Our results show that the detected antigens appear to be algal analogs of PCNA.     

ACTIVE GROWTH OF THE OCEANIC DINOFLAGELLATE CERATIUM TERES IN THE CARIBBEAN AND SARGASSO SEAS ESTIMATED BY CELL CYCLE ANALYSIS1

The growth rate of an oceanic dinoflagellate, Ceratium teres Kofoid, was investigated in the Sargasso and Caribbean Seas from September 1989 to July 1990 using the cell cycle analysis method. Estimated growth rates ranged from 0.29 to 0.58 day^?1 and were 1.5–7.2 times higher than generally accepted rates for oceanic dinoflagellates. The higher rates in this report were mainly due to an improvement in techniques that determine the duration of a terminal cell cycle phase in situ. The day-to-day variation in growth rates was surprisingly small, but, from long-term measurements, a weak correlation was found among temperature, daily irradiance, and seasonal growth rate. The calculated species-specific primary production ranged from 0.5 to 1.8 mg C·m^?2·day^?1, about 1% of the estimated total production. Ceratium teres may be an important carbon source at the base of the grazing food chain.     

INFLUENCE OF CELL VOLUME ON THE GROWTH AND SIZE REDUCTION OF MARINE AND ESTUARINE DIATOMS1

The maximal growth rate (μ_max) of 19 marine and estuarine diatoms decreased with increasing cell volume (V). The relationship between log μ_max (Y) and log V (X) was calculated. Statistical analyses showed that the slope of the equation was not significantly different from those obtained by other researchers and that the 95% confidence intervals of mean μ_max at cell volumes of 10³–10⁵μm³ were not significantly different from those cited in most studies. A new regression line for diatoms was calculated as follows: log μ_max= 0.47–0.14 log V; r =–0.69. The rate of size reduction per generation of the 19 diatom species ranged from 0.03 to 0.87 μm per generation. The rate increased with increasing cell length and cell volume and with decreasing maximum division rate. Statistical analyses showed that the rate was closely related to the cell volume and to the reciprocal of the growth rate. The relationships between maximal growth rate and cell volume and between rate of size reduction and cell volume showed that a diatom with a large volume had a smaller maximal growth rate and a larger rate of size reduction than a diatom with a small volume. The estimates using the equation for the regression line between the rate of size reduction and the reciprocal of maximum division rate indicated that a diatom with a high maximum division rate would need more generation equivalents for a certain size reduction than a diatom with a low maximum division rate, but the periods required for reduction would be approximately equal irrespective of maximum division rate.     

MOLECULAR CLONING AND EXPRESSION OF THE PROLIFERATING CELL NUCLEAR ANTIGEN GENE FROM THE COCCOLITHOPHORID PLEUROCHRYSIS CARTERAE (HAPTOPHYCEAE)1

The gene encoding proliferating cell nuclear antigen (PCNA) was isolated from the marine coccolithophorid microalga Pleurochrysis carterae (Braarud et Fagerland) Christensen (Haptophyceae). Two mRNAs (Pcpcna1 and Pcpcna2) were identified and contained an identical coding region for 222 amino acid residues and an untranslated sequence of 302 base pair (Ut1) and 246 base pair (Ut2), respectively. Comparison between PCR‐derived genomic DNA fragments and cDNA sequences revealed five introns. The coding region of Pcpcna is similar to counterparts in other organisms and contains highly conserved functional domains. Phylogenetic analyses indicated clustering of Pcpcna with pcna in its haptophyte relative Isochrysis galbana Parke. A recombinant fusion protein of Pcpcna, overexpressed in Escherichia coli, was recognized by the PC10 antibody against rat PCNA. Using RT‐PCR and Western blotting, Pcpcna was found to be highly transcribed and translated during the exponential growth phase relative to the stationary growth phase, with a positive correlation between gene expression and growth rate. It can be concluded that the pcna is conserved in this coccolithophorid phytoplankton and that its expression is growth stage related.     

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.     

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.     

TARGETING AND COVALENT MODIFICATION OF CELL WALL AND MEMBRANE PROTEINS HETEROLOGOUSLY EXPRESSED IN THE DIATOM CYLINDROTHECA FUSIFORMIS (BACILLARIOPHYCEAE)

Diatoms are unicellular organisms encased by silica-based cell walls that display species-specific structures. Morphogenesis of diatom cell walls is believed to be controlled by a polysaccharide/protein-matrix that remains associated with mature cell walls. Recently, a family of calcium-binding glycoproteins, the frustulins, has been identified as major diatom cell wall component. Here we describe a transformation-based approach to investigate intracellular targeting and function of frustulins. When ε-frustulin from the diatom Navicula pelliculosa is expressed in Cylindrotheca fusiformis, it is correctly targeted into the cell wall. Furthermore, the unique N-terminus of ε-frustulin was properly modified, indicating that C. fusiformis and N. pelliculosa contain homologous frustulin-processing proteases. In a different transformation experiment, a modified version of the Chlorella kessleri hexose/H⁺ symporter bearing a bacterial biotinyl-acceptor domain was expressed in C. fusiformis. The transporter became biotinylated in vivo and was functionally incorporated into the plasma membrane, allowing C. fusiformis to take up ¹⁴C-glucose and ¹⁴C-glucosamine. Stage-specific radioactive labeling with this transformant revealed that secretion of frustulins is strongly enhanced during cell wall development. The data presented in this study demonstrate for the first time functional expression of a membrane protein and correct targeting of a cell wall protein heterologously expressed in a diatom cell.     

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.     

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.     

EFFECT OF IRRADIANCE ON GROWTH AND REPRODUCTIVE PROCESSES DURING THE CELL CYCLE IN SCENEDESMUS ARMATUS (CHLOROPHYTA)1

Synchronized populations of the chlorococcal alga Scenedesmus armatus (Chod.) Chod. were grown under five irradiance levels. During the cell cycles of these populations, reproductive processes such as DNA replication, nuclear division, protoplast fission, and daughter cell release and growth processes such as RNA and protein accumulation were followed. The amount of RNA and proteins increased stepwise with a short time interval between individual steps during which the rate of RNA and protein accumulation decreased. At each of the steps, the amount of RNA and protein approximately doubled and the number of steps increased with irradiance. At the end of each of the growth steps, a commitment to trigger the sequence of reproductive events (DNA replication, nuclear division, protoplast fission) was attained. After attaining the commitment point, the cells were able to trigger and terminate the whole reproductive sequence without any further growth, that is, even in the dark when the external supply of energy was cut off. With increasing irradiance, the number of commitment points attained during one cell cycle increased from one to four. Consequently, one to four sequences of the reproductive steps were triggered, and each of them ended by doubling the reproductive structures, which resulted in the formation of 2, 4, 8, or 16 daughter cells. The length of the precommitment periods shortened with increasing irradiance as the result of an increasing rate in growth. The length of postcommitment periods showed light independence and remained constant at the range of irradiances at which the number of growth steps and, consequently, the number of sequences of reproductive events did not change. At higher irradiances, the number of sequences of reproductive events increased, which caused a prolongation of postcommitment periods. The length of the cell cycle varied as a result of this distinct effect of irradiance on pre- and postcommitment periods.     

BIOLOGICAL AND CHEMICAL CHARACTERISTICS OF THE GIANT DIATOM ETHMODISCUS (BACILLARIOPHYCEAE) IN THE CENTRAL NORTH PACIFIC GYRE

Cells of the giant diatom Ethmodiscus Castr. gathered from the upper 15 m were examined for O₂ evolution, nitrate reductase activity (NRA), C and N composition, internal NO concentrations, , and ¹⁵NO, ¹⁵NH , and ³²Si uptake in a series of cruises in the central N. Pacific gyre. The δ¹⁵N (2.56–5.09 ‰), internal NO concentrations (0.0– 11.5 mM NO⁻), and NRA (6.7 ± 4.7 × 10⁻⁴μM NO cell ⁻¹·h⁻¹) were consistent with recent exposure to elevated nitrate concentrations and utilization of deep NO as a primary N source. These results are similar to other diatoms that migrate vertically to the nutricline as part of their life cycle. Rate measures (Si[OH]₄ uptake, NRA, and O₂ evolution) indicated surface doubling times from 45 h to 75 h. Both NO and NH uptake in surface waters were low and inadequate to supply N needs at surface NO and NH concentrations. Our results suggest a partitioning in nutrient acquisition, with N acquired at depth and C and Si acquired at the surface. Doubling rates were two to three times higher than predicted from cell volume and C content models. These data are consistent with the observed elemental content being lower than expected because of the dominance of cell volume by the vacuole. Our calculations suggest that Ethmodiscus contributes little to the biogeochemistry of the upper water column via upward nutrient transport. Although reported as a paleo-upwelling indicator, thisevidence suggests that Ethmodiscus has adapted to the nutrient-poor open ocean by a vertical migration strategy and has biological characteristics inconsistent with a upwelling indicator.     

THE NUCLEAR CELL CYCLE IN CHLAMYDOMONAS (CHLOROPHYCEAE)1

The timing of replication and division of the Chlamydomonas Ehrenberg nucleus in the vegetative cell cycle and at gametogenesis was examined, using fluorescence microspectrophotometry with two fluorochromes, mithramycin and 4′,6-diamidino-2-phenylindole (DAPI). Under appropriate conditions, these bind specifically to DNA, and the fluorescence of the DNA fluorochrome complex is a quantitative measure of the DNA content. The alga is a haplont, which produces 2ⁿ daughter cells at the time of vegetative reproduction; cytokinesis and daughter cell release lag behind karyokinesis. No nucleus was found to contain more than the 2c quantity of DNA. Hence daughter cell production proceeds by doubling of the nuclear DNA followed by karyokinesis, in a repetitive sequence. As reported previously for C. reinhardtii Dangeard, the gametes of C. moewusii Gerloff contain the 1c amount of nuclear DNA. Several conflicting interpretations of the cell cycle sequence proposed in the literature were resolved.     

THE NATURAL LIFE CYCLE IN WILD POPULATIONS OF DIATOMA MONILIFORMIS (BACILLARIOPHYCEAE) AND ITS DISRUPTION IN AN ABERRANT ENVIRONMENT1

We studied how size variation in populations of Diatoma moniliformis Kütz. was influenced by environmental effects on the diatom life cycle. One of the two populations sampled monthly in the northern Baltic Sea grew under natural conditions; the other population was in a cooling water discharge channel of a nuclear power plant, where the temperature and flow rate of the water were artificially higher. The life cycle was synchronous at the natural site, with sexual reproduction occurring in the winter; most of the initial cells were found in March-April. After this, a reduction in cell size occurred, and the vegetative life cycle consisted of two parts. During the first part, cell volume decreased, whereas the surface area to volume ratio increased, and during the second part of the cycle, both of these parameters decreased. No direct evidence was found for the existence of a supra-annual life cycle in D. moniliformis, as convincing modes fm large cells were lacking in the size-frequency distributions. It was concluded from extrapolations of the data that the natural life cycle of D. moniliformis probably lasts 2 or 3 years. The changes in cell proportions during the life cycle fit well with annual growth cycles of D. moniliformis at the natural site (i.e. the cells had high surface area to volume ratios during the period of optimal growth in late spring [May-June]). At the site affected by cooling water discharge, the synchronization of the natural life cycle was disrupted, but some seasonal size variation did occur. Under natural conditions, auxosporulation is probably triggered by a combination of small cell size, low water temperature (0–3° C), and rapidly increasing light intensity or daylength in late winter to early spring. When these conditions were not met (e.g. at the heated site, the required low temperature was absent), auxosporulation did not occur simultaneously. This paper also presents scanning electron photomicrographs showing the typical shape and fine structure of the initial cell of D. moniliformis. These cells are semispherical in cross section, possess a pronounced curvature along the longitudinal axis, and are bent in the perualvar plane.     

CHANGE OF UBIQUITINATED PROTEINS DURING THE CELL CYCLE IN CHLAMYDOMONAS (CHLOROPHYTA)1

Changes in the amount of heat shock-related ubiquitinated proteins in Chlamydomonas were investigated during the cell cycle and gamete induction. In a division-synchronized culture induced by periodic illumination, the amount of the 28-kDa ubiquitinated protein increased during the dark phase. This increase correlated with the increase of total DNA. Such an increase was repressed when nuclear DNA replication was inhibited with aphidicolin. These results suggest that ubiquitination to form the 28-kDa protein is involved in nuclear DNA replication or during the cell cycle. The amount of 31-kDa ubiquitinated protein gradually increased throughout the light phase and decreased in the dark phase. The amount of 28-kDa ubiquitinated protein also increased during gamete induction caused by nitrogen starvation, while that of the 31-kDa did not. These results suggest that the change of ubiquitination of 28-kDa protein mat play a fundamental role in the cell cycle and gamete induction in Chlamydomonas.     

DISTRIBUTION OF TWO TYPES OF EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) IN THE NORTHEAST ATLANTIC REGION AS DETERMINED BY IMMUNOFLUORESCENCE AND COCCOLITH MORPHOLOGY1

Culture strains of Emiliania huxleyi (Lohmann 1902) Hay et al. 1967 were placed into two groups designated E. huxleyi type A and type B on the basis of coccolith morphology and immunological properties of the coccolith polysaccharide. We studied the distribution of these types in the North Atlantic region using an indirect immunofluorescence assay with antisera directed against the coccolith polysaccharide of E. huxleyi type A and type B and epifluorescence microscopy. In field samples taken in the Northeast Atlantic Ocean, E. huxleyi type A was found exclusively. In contrast, type B was dominant in the North Sea. Scanning electron microscopy of the samples revealed the same unequal distribution of the two types as found with the immunofluorescent-labelling assay.     

SHORT-TERM RESPONSE OF THE DIADINOXANTHIN CYCLE AND FLUORESCENCE YIELD TO HIGH IRRADIANCE IN CHAETOCEROS MUELLERI (BACILLARIOPHYCEAE)1

The relationship between the diadinoxanthin cycle and changes in fluorescence yield in the diatom Chaetoceros muelleri Lemm. (clone CH10, Amorient Aquafarm, Inc., Hawaii) was investigated. High-light-induced changes in fluorescence yield and xanthophyll de-epoxidation occurred very rapidly (first order rate constant 1.60 min^?1). The observed light-induced changes in diatoxanthin and diadinoxanthin concentration were consistent with a two-pool scheme for diadinoxanthin, one of which does not undergo de-epoxidation. Changes in xanthophyll concentration correlated with changes in in vivo absorbance indicating that diadinoxanthin cycle activity in vivo can be monitored spectrophotometrically. However, changes in cell absorbance were small relative to total optical absorption cross section. Increases in the concentration of diatoxanthin were linearly correlated with increases in the rate constant for thermal de-excitation in the antenna of photosystem II (PSII). Antenna quenching produced or mediated by diatoxanthin may, thus, protect the PSII reaction center in diatoms. Changes in the maximum fluorescence yield suggested that changes in the reaction center also contributed to nonphotochemical quenching of fluorescence. Thus, reaction center quenching affected the relationship between antenna quenching and changes in photochemical efficiency producing the effect of a decrease in fluorescence yield without a decrease in photochemical efficiency.     

RELATIONSHIPS BETWEEN NITRATE REDUCTASE ACTIVITY AND RATES OF GROWTH AND NITRATE INCORPORATION UNDER STEADY-STATE LIGHT OR NITRATE LIMITATION IN THE MARINE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1

Although activity of the enzyme nitrate reductase (NR) can potentially be used to predict the rate of nitrate incorporation in field assemblages of marine phytoplankton, application of this index has met with little success because the relationship between the two rates is not well established under steady-state conditions. To provide a basis for using NR activity measurements, the relationships among NR activity, growth rate, cell composition, and nitrate incorporation rate were examined in cultures of Thalassiosira pseudonana (Hustedt)Hasle and Heimdal, growing a) under steady-state light limitation, b) during transitions between low and high irradiance (15 or 90 μmol quanta.m^?2.s^?1), and c) under steady-state nitrate limitation. Using a modified assay for NR involving additions of bovine serum albumin to stabilize enzyme activity, NR activity in light-limited cultures was positively and quantitatively related to calculated rates of nitrate incorporation, even in cultures that were apparently starved of selenium. During transitions in irradiance, growth rates acclimated to new conditions within 1 day; through the transition, the relationship between NR activity and nitrate incorporation rate remained quantitative. In nitrate-limited chemostat cultures, NR activity was positively correlated with growth rate and with nitrate incorporation rates, but the relationship was not quantitative. NR activity exceeded nitrate incorporation rates at lower growth rates (<25% of nutrient-replete growth rates), but chemostats operating at such low dilution rates may not represent ecologically relevant conditions for marine diatoms. The strong relationship between NR activity and nitrate incorporation provides support for the idea that NR is rate-limiting for nitrate incorporation or is closely coupled to the rate-limiting step. In an effort to determine a suitable variable for scaling NR activity, relationships between different cell components and growth rate were examined. These relationships differed depending on the limiting factor. For example, under light limitation, cell volume and cell carbon content increased significantly with increased growth rate, while under nitrate limitation cell volume and carbon content decreased as growth rates increased. Despite the differences found between cell composition and growth rate under light and nitrate limitation, the relationships between NR activity scaled to different compositional variables and growth rate did not differ between the limitations. In field situations where cell numbers are not easily determined, scaling NR activity to particulate nitrogen content may be the best alternative. These results establish a strong basis for pursuing NR activity measurements as indices of nitrate incorporation in the field.     

EFFECT OF TEMPERATURE ON LIGHT-LIMITED GROWTH AND CHEMICAL COMPOSITION OF SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)1

Phytoplankton growth rate in response to irradiance can be approximated by a hyperbola defined by three coefficients: i) initial slope (α); ii) asymptote (μ_m); and, iii) X-axis intercept or compensation irradiance (I_c). To mathematically represent the interaction of temperature and irradiance on growth rate, one must describe the relationship between these constants and temperature. The marine diatom, Skeletonema costatum (Greville) Cleve, was grown in unialgal culture at different levels of irradiance and 2-3 photoperiods at 0, 5, 10, 16 and 22 C. The value of I_c is ca. 1.0 ly·day^?1 or less at all temperatures. The initial slope (div·ly^?1) is a “u-shaped” function of temperature described by the second degree polynomial, α= 0.25–0.02T+0.001T². Within the range 0–10 C, μ_m (div·day^?1) is an exponential function of temperature described by the equation, μ_m= 0.48 exp (0.126T). At each temperature and selected levels of irradiance, cell size and cellular content of C, N and chl a were determined. The C:chl a and N:chl a ratios increased with irradiance because of increases in C and decreases in chl a. At lower temperatures (0, 5, 10 C), the rate of increase of both ratios with irradiance was greater than at the higher temperatures (16, 22 C). Cellular content of N was independent of irradiance and temperature, and the C:N ratio ranged from 5 to 8 with a slight tendency to lower values at low irradiance. Cell volume was not influenced by either temperature or irradiance.