AMMONIUM INDUCED PHOTOSYNTHETIC SUPPRESSION IN AMMONIUM LIMITED DUNALIELLA TERTIOLECTA (CHLOROPHYTA)1

Dunaliella tertiolecta (Butcher) was grown in chemostat culture over a wide range of ammonium limited growth rates. The addition of ammonium caused a rapid temporary suppression of photosynthetic carbon fixation. The magnitude of ammonium induced photosynthetic suppression increased with the severity of ammonium limitation. Cells growing at rates greater than ca. 80%μ_max exhibited no photosynthetic suppression in response to additions of the limiting nutrient. The duration of photosynthetic suppression was related to the concentration of added ammonium. Immediately following the suppression, photosynthesis was enhanced with respect to the controls. The eventual degree of enhancement increased with the concentration of added ammonium. Steady-state cellular chlorophyll concentrations, photosynthetic rates, and assimilation numbers are reported.     

PHOTOSYNTHETIC FUNCTION IN DUNALIELLA TERTIOLECTA (CHLOROPHYTA) DURING A NITROGEN STARVATION AND RECOVERY CYCLE

Phytoplankton can be exposed to periods of N starvation with episodic N resupply. N starvation in Dunaliella tertiolecta (Butcher) measured over 4 days was characterized by slow reduction in cell chl and protein content and chl/carotenoid ratio and a decline in photosynthetic capacity and maximum quantum yield of photosynthesis (F_v/F_m). In the early stages of N starvation, cell division was maintained despite reduction in cellular chl. Chl content was more sensitive than carotenoids to N deprivation, and cellular chl a was maintained preferentially over chl b under N starvation. NO₃^? resupply stimulated rapid and complete recovery of F_v/F_m (from 0.4 to 0.7) within 24 h and commencement of cell division after 10 h, although N‐replete levels of cell chl and protein were not reestablished within 24 h. Recovery of F_v/F_m was correlated with increases in cell chl and protein and was more related to increases in F_m than to changes in F₀. Recovery of F_v/F_m was biphasic with a second phase of recovery commencing 4–6 h after resupply of NO₃^?. Uptake of NO₃^? from the external medium and the recovery of F_v/F_m, cell chl, and protein were inhibited when either cytosolic or chloroplastic protein synthesis was inhibited by cycloheximide or lincomycin, respectively; a time lag observed before maximum NO₃^? uptake was consistent with synthesis of NO₃^? transporters and assimilation enzymes. When both chloroplastic and cytosolic translation was inhibited, F_v/F_m declined dramatically. Dunaliella tertiolecta demonstrated a capacity to rapidly reestablish photosynthetic function and initiate cell division after N resupply, an important strategy in competing for limiting inorganic N resources.     

INTERACTIONS AMONG PHOSPHATE UPTAKE,PHOTOSYNTHESIS, AND CHLOROPHYLL FLUORESCENCE IN NUTRIENT‐LIMITED CULTURES OF THE CHLOROPHYTE MICROALGA DUNALIELLA TERTIOLECTA1

Phosphate‐limited and phosphate‐sufficient continuous cultures of the marine chlorophyte microalga Dunaliella tertiolecta Butcher were examined for their responses to the addition of phosphate. Phosphate‐limited cultures showed a marked quenching of chl fluorescence following a pulse of phosphate. This response was absent from cells growing under phosphate‐sufficient conditions. Both the extent of fluorescence quenching (where present) and the initial rate of change in quenching were dependent on the concentration of phosphate added to cell suspensions and on the degree of limitation (growth rate in continuous culture). The addition of phosphate also brought about a transient decrease in photosynthetic oxygen evolution and a stimulation in respiration, which were relaxed as the added phosphate was depleted from the external medium. The applicability of using nutrient‐induced fluorescence transients as a tool to identify the nutrient status of phytoplankton populations is discussed.     

NITROGEN LIMITATION IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE) LEADS TO INCREASED SUSCEPTIBILITY TO DAMAGE BY ULTRAVIOLET‐B RADIATION BUT ALSO INCREASED REPAIR CAPACITY1

Chl fluorescence was used to measure the photosynthetic capacity of the green alga Dunaliella tertiolecta in order to investigate interactions between susceptibility to acute UV‐B radiation (UVBR, 280–320 nm) exposure and decreased nitrogen availability. Under UVBR exposure the decline in the fluorescence parameters F_v/F_m (the maximum effective quantum yield Φ_PSIIe‐max) and F_v′/F_m′ (the operational quantum yield of PSII, Φ_PSIIe) were enhanced with higher UVBR fluxes, with the data well described by the Kok model, inferring that a dynamic balance existed between damage and repair with the repair proportional to the pool size of inactivated targets. When UVBR exposure was coupled with nitrogen limitation, the inhibition of photosynthesis was intensified. Under the more severely N‐limited conditions, the damage rate increased. Unexpectedly, repair rates were also stimulated under N‐limited conditions, although this was insufficient to counteract the increase in damage, so the overall effect of N limitation was an enhancement of UVBR‐induced inhibition of photosynthesis.     

STATE TRANSITIONS AND NONPHOTOCHEMICAL QUENCHING DURING A NUTRIENT‐INDUCED FLUORESCENCE TRANSIENT IN PHOSPHORUS‐STARVED DUNALIELLA TERTIOLECTA1

Assessments of nutrient‐limitation in microalgae using chl a fluorescence have revealed that nitrogen and phosphorus depletion can be detected as a change in chl a fluorescence signal when nutrient‐starved algae are resupplied with the limiting nutrient. This photokinetic phenomenon is known as a nutrient‐induced fluorescence transient, or NIFT. Cultures of the unicellular marine chlorophyte Dunaliella tertiolecta Butcher were grown under phosphate starvation to investigate the photophysiological mechanism behind the NIFT response. A combination of low temperature (77 K) fluorescence, photosynthetic inhibitors, and nonphotochemical quenching analyses were used to determine that the NIFT response is associated with changes in energy distribution between PSI and PSII and light‐stress‐induced nonphotochemical quenching (NPQ). Previous studies point to state transitions as the likely mechanism behind the NIFT response; however, our results show that state transitions are not solely responsible for this phenomenon. This study shows that an interaction of at least two physiological processes is involved in the rapid quenching of chl a fluorescence observed in P‐starved D. tertiolecta: (1) state transitions to provide the nutrient‐deficient cell with metabolic energy for inorganic phosphate (P_i)‐uptake and (2) energy‐dependent quenching to allow the nutrient‐stressed cell to avoid photodamage from excess light energy during nutrient uptake.     

PHYSIOLOGICAL RESPONSES TO PHOSPHORUS LIMITATION IN BATCH AND STEADY-STATE CULTURES OF DUNALIELLA TERTIOLECTA (CHLOROPHYTA): A UNIQUE STRESS PROTEIN AS AN INDICATOR OF PHOSPHATE DEFICIENCY1

A protein unique to phosphorus stress observed in Dunaliella tertiolecta Butcher was studied in the context of phosphate-limited cell physiology and is a potential diagnostic indicator of phosphate deficiency in this alga. Cells were grown over a range of limited, steady-state growth rates and at maximum (replete) and zero (phosphate-starved) growth rates. The stress protein, absent in nutrient-replete cells, was produced under all steady-state phosphate-limited conditions and increased in abundance with increasing limitation (decreasing growth rate). Cellular carbon: phosphorus ratios and the maximum uptake rate of phosphate (V_m) increased with increasing limitation, whereas the ratio of chlorophyll a: carbon decreased. Alkaline phosphatase activity did not respond to limitation but was measurable in starved, stationary-phase cells. F_v/F_m, a measure of photochemical efficiency, was a nonlinear, saturating function of p, as commonly observed under N limitation. The maximum F_v/F_m of 0.64 was measured in nutrient-replete cells growing at μ_max, and a value of zero was measured in stationary-phase starved cells. When physiological parameters were compared, the P-stress protein abundance and F_v/F_m were the most sensitive indicators of the level of deficiency. The stress protein was not produced under N- or Fe-limited conditions. It is of high molecular weight (>200) and is associated with internal cell membranes. The stress protein has several characteristics that make it a potential diagnostic indicator: it is 1) unique to phosphorus limitation (i.e. absent under all other conditions), 2) present under limiting as well as starved conditions, 3) sensitive to the level of limitation, and 4) observable without time-course incubation of live samples.     

ASSESSING PHYSIOLOGICAL STRESS IN THALASSIOSIRA FLUVIATILIS (BACILLARIOPHYTA) AND DUNALIELLA TERTIOLECTA (CHLOROPHYTA) WITH DCMU-ENHANCED FLUORESCENCE1

Exponentially growing cultures of Thalassiosira fluviatilis Hustedt and Dunaliella tertiolecta Butcher were exposed to 4 min temperature shocks of 5° to 20°C above ambient (20°C). Photosynthetic carbon fixation, changes in in vivo fluorescence and fluorescence on the addition of the herbicide DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) were measured over the subsequent 24 h. The fluorescence ratio (R, DCMU-enhanced fluorescence/in vivo fluorescence) paralleled changes in photosynthesis over this period; both were significantly reduced (P < 0.05) by temperature shocks of +15° and +20° C, but +5° and +10° C treatments had no inhibitory effect on either relative to the control. The instantaneous response obtained with the fluorescence ratio indicates that the technique might be applicable to routine bioassay procedures and thus replace the time consuming methods now used for the estimation of ¹⁴C-incorporation and growth rates.     

PHOTORESPIRATION IN CONTINUOUS CULTURE OF DUNALIELLA TERTIOLECTA (CHLOROPHYTA): RELATIONSHIPS BETWEEN SERINE, GLYCINE, AND EXTRACELLULAR GLYCOLATE

The concentrations of extracellular glycolate and intracellular free pools of serine and glycine were monitored in nitrogen-limited continuous cultures of Dunaliella tertiolecta (Butcher) UTEX LB999, grown at two different irradiances on a light:dark cycle. Under steady-state conditions, this microalga excreted into the medium a large amount of glycolate during the light phase, up to 100 nmol·(10⁶ cells)⁻¹ for a cell concentration of around 1.5 10⁸ cells·L⁻¹, but glycolate disappeared from the dissolved phase in the dark. Cells grown at 70 and those grown at 430 μmol photons·m⁻²·s⁻¹ differed in maximal glycolate concentration, intracellular serine and glycine concentrations, and serine:glycine ratio. Reversal of these photon flux densities to which the cultures were exposed caused rapid modification of the extracellular glycolate and intracellular serine and glycine pools. These results suggest that photorespiratory metabolism in D. tertiolecta could be approximately quantified by measuring the changes in dissolved glycolate and intracellular serine and glycine concentrations, extending previous results from cultured phytoplankton and suggesting methods for field studies.     

EFFECTS OF TEMPERATURE ON GROWTH,LIGHT ABSORPTION,AND QUANTUM YIELD IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

The effects of growth temperature on the marine chlorophyte Dunaliella tertiolecta Butcher were studied to provide a more mechanistic understanding of the role of environmental factors in regulating bio-optical properties of phytoplankton. Specific attention was focused on quantities that are relevant for modeling of growth and photosynthesis. Characteristics including chlorophyll a (chl z)-specific light absorption (a*_ph(λ)), C:chl a ratio, and quantum yield for growth (φ_μ) varied as functions of temperature under conditions of excess light and nutrients. As temperature increased over the range examined (12°-28°C), intracellular concentrations of chl a increased by a factor of 2 and a*_ph(λ) values decreased by more than 50% at blue to green wavelengths. The lower values of a*_ph(λ) were due to both a decrease in the abundance of accessory pigments relative to chl a and an increase in pigment package effects arising from higher intracellular pigment concentrations. Intracellular pigment concentration increased as a consequence of higher cellular pigment quotas combined with lower cell volume. At high growth temperatures, slightly more light was absorbed on a per-cell-C basis, but the dramatic increases in growth rate from μ= 0.5 d^?1 at 12° C to μ= 2.2 d^?1 at 28°C were primarily due to an increase in φ_μ (0.015–0.041 mol C (mol quanta)^?1). By comparison with previous work on this species, we conclude the effects of temperature on a*_ph(λ) and φ_μ are comparable to those observed for light and nutrient limitation. Patterns of variability in a*_ph(λ)and φ_μ as a function of growth rate at different temperatures are similar to those previously documented for this species grown at the same irradiance but under a range of nitrogen-limited conditions. These results are discussed in the context of implications for bio-optical modeling of aquatic primary production by phytoplankton.     

EFFECTS OF LONG TERM EXPOSURE TO LOW TEMPERATURE ON THE PHOTOSYNTHETIC APPARATUS OF DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

The effects of long term exposure to suboptimal growth temperature on the photosynthetic apparatus of Dunaliella tertiolecta Butcher were investigated using carbon fixation rate versus irradiance curves and the variable fluorescence induction method. Carbon fixation rates per unite chlorophyll a at saturating (p^B_m) and subsaturating (α^B) irradiances were 55% and 39% lower, respectively, at 12° C than at 20° C. Chlorophyll a quotas and the spectrally averaged in vivo absorption cross section normalized to chlorophyll a (a^*) were not significantly different at these two temperatures. Analysis of the fluorescence kinetics revealed 1) no significant variations of the amount of PSII photoactive reaction centers per unit chlorophyll a, 2) a 14% decrease of the PSII quantum yield(+) and 3) a 29% decrease of the energy transfer efficiency between the light harvesting chlorophyll a pigment bed and the PSII reaction centers. The decrease in energy transfer efficiency between the antennae and the PSII reaction centers at 12° C was interpreted as a mechanism to avoid photoinhibition.     

INTERACTIONS BETWEEN UV‐B EXPOSURE AND PHOSPHORUS NUTRITION. I. EFFECTS ON GROWTH,PHOSPHATE UPTAKE,AND CHLOROPHYLL FLUORESCENCE1

The interactive effects of P starvation and exposure to UV radiation on growth rates, quantum efficiency of PSII electron transport, and P‐uptake capacity of the chlorophyte microalga Dunaliella tertiolecta Butcher are presented. Ultraviolet radiation did not in itself cause marked changes in growth rate, though it did induce changes in the effective quantum yield of PSII. Depriving cells of phosphate resulted in significant changes in all parameters examined. The decline of growth rate and fluorescence parameters after P starvation was significantly faster in the presence of UV radiation. Ultraviolet radiation also stimulated the magnitude of the transient changes in chl fluorescence (nutrient‐induced fluorescence transient) exhibited by P‐starved cells after resupply of that nutrient.     

PHOTOADAPTATION AND THE “PACKAGE” EFFECT IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

In the marine unicellular chlorophyte, Dunaliella tertiolecta Butcher, the spectrally averaged m vivo absorption cross section, normalized to chlorophyll a (so-called a* values), vary two-fold in response to changes in growth irradiance. We used a kinetic approach to examine the specific factors which account for these changes in optical properties as cells photoadapt. Using Triton X-100 to solubilize membranes, we were able to differentiate between “package” effects and pigmentation effects. Our analyses suggest that 43–49% of the variability in a* is due to changes in pigmentation, whereas 51–57% is due to the “package” effect. Further analyses revealed that changes in cell sue did not significantly affect packaging, while thylakoid stacking and the transparency of thylakoid membranes were important factors. Our results suggest that thylakoid membrane protein/lipid ratios change during photoadaptation, and these changes influence the effective rate of light harvesting per unit chlorophyll a.     

ORGANIC CARBON RELEASE BY DUNALIELLA SALINA (CHLOROPHYTA) UNDER DIFFERENT GROWTH CONDITIONS OF CO2, NITROGEN,AND SALINITY1

Two strains of Dunaliella salina (Dunal) Teod., UTEX 1644 and UTEX 200, were cultured under different growth regimes, including 10 mM NO₃^? or NH₄⁺, 1.5 or 3.0 M NaCl, and low (0.035%) or high (5%) CO₂ in air. The release of ¹⁴C-labeled dissolved organic carbon (DOC), expressed as a rate and as a percentage of photosynthetic ¹⁴CO₂ assimilation, was subsequently determined. The percentage of DOC released was inversely related to cell density in the assay medium, but photosynthesis on a per-cell basis was not. Release of DOC was low, in the range of 1–5% of photosynthesis, but during acclimation to growth on NH₄⁺, it rose to 11%. The presence of NH₄⁺ rather than NO₃^? in the growth medium increased the rate of release by both strains, but the percentage release was stimulated only in UTEX 200 cells, because their photosynthetic rate was depressed by NH₄⁺. For UTEX 1644, high, as compared to low, CO₂-grown cells, had somewhat higher rates and percentages of DOC release, but release from UTEX 200 cells was unaffected by the growth-CO₂. The rate of DOC release by high CO₂-grown cells was not enhanced at a low concentration of dissolved inorganic carbon, indicating that the released material did not originate from the photorespiratory pathway. The effects of NaCl on DOC release varied with strain and growth conditions. For UTEX 200, the cells in NO₃^?, but not NH₄⁺, exhibited a doubling or more in percentage of release with a doubling in NaCl concentration, irrespective of growth-CO₂. With UTEX 1644 the low CO₂-grown cells showed the greatest enhancement in 3.0 M NaCl. Organic matter accumulated on the external surface of the cell membrane and constituted a well-defined cell-coat, which was more dense in NH₄⁺ than in NO₃^?-grown cells. Microtubules, which may play a role in maintaining cell shape, were observed just below the plasma membrane. From a practical viewpoint, the presence of organic material in the hypersaline ponds of salt-works is detrimental to salt production. When D. salina cells become abundant in such ponds, the attendant, continuous release of DOC may make a significant contribution to the problem.     

INHIBITION OF CASPASE‐LIKE ACTIVITIES PREVENTS THE APPEARANCE OF REACTIVE OXYGEN SPECIES AND DARK‐INDUCED APOPTOSIS IN THE UNICELLULAR CHLOROPHYTE DUNALIELLA TERTIOLECTA1

When the chlorophyte alga Dunaliella tertiolecta Butcher is placed in darkness, a form of programmed cell death with many similarities to apoptosis is induced, including the induction of caspase‐like proteases. Many uncertainties about the regulation and mediators that participate in the process remain. To examine the relationship between caspase‐like activities and different apoptotic events (i.e., phosphatidylserine [PS] translocation), increases in membrane permeability and numbers of dead cells revealed by SYTOX‐green staining, and the generation of reactive oxygen species (ROS), we used the broad‐range caspase inhibitor Boc‐D‐FMK to block the activity of the whole class of caspase‐like proteins simultaneously. In the presence of the inhibitor, ROS were not produced, and cells did not die. Loss of membrane asymmetry, indicated by external labeling of PS by annexin V, was apparent at midstages of light deprivation, although it did not conform to the typical pattern for PS exposure observed in metazoans or vascular plants, which occurs at early stages of the apoptotic event. Thus, we have evidence for a link between ROS and cell death involving caspase‐like enzymes in an alga. The fact that caspase‐like inhibitors prevent not only cell death, but also ROS and loss of cell membrane integrity and asymmetry, suggests that caspase‐like proteases might have regulatory roles early in cell death, in addition to dismantling functions.     

LINKING TIME‐DEPENDENT CARBON‐FIXATION EFFICIENCIES IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE) TO UNDERLYING METABOLIC PATHWAYS1

The chl‐specific short‐term ¹⁴C‐based production (P^b) measurement is a widely used tool to understand phytoplankton responses to environmental stresses. However, among the metabolic consequences of these stresses is variability in lifetimes of newly fixed carbon that cause P^b to range between chl‐specific net primary production (NPP*) and chl‐specific gross photosynthetic electron flow that is available for carbon reduction () depending on growth rate. To investigate the basis for this discrepancy, photosynthate utilization was characterized in Dunaliella tertiolecta Butcher grown at three different growth rates in N‐limited chemostats. P^b was measured throughout a 2 min to 24 h time course and showed clear growth‐rate‐dependent differences in lifetimes of newly fixed carbon. ¹⁴C pulse‐chase experiments revealed differences in patterns of carbon utilization between growth rates. At high growth rate, the majority of ¹⁴C was initially fixed into polysaccharide and lipid, but the relative contribution of each labeled biochemical pool to the total label changed over 24 h. In fast‐growing cells, labeled polysaccharides decreased 50%, while labeled lipids increased over the first 4 h. At low growth rate, ¹⁴C was initially incorporated primarily into protein, but the contribution of labeled protein to the total label increased over the next 24 h. Together, time‐resolved measurements of P^b and cellular NAD and NADP content suggest an enhanced role for alternative dissipation pathways at very low growth rate. Findings of this study contribute to an integrated understanding of growth‐rate‐dependent shifts in metabolic processes from photosynthesis to net growth.     

MOLECULAR CHARACTERIZATION AND ANTIBODY DETECTION OF A NITROGEN‐REGULATED CELL‐SURFACE PROTEIN OF THE COCCOLITHOPHORE EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE)1

Dissolved organic nitrogen (DON) can account for a significant portion of total nitrogen in some aquatic environments, and many species of phytoplankton are able to scavenge nitrogen from this pool especially when inorganic nitrogen is limiting. Emiliania huxleyi (Lohmann) H. W. Hay et H. Mohler is able to use various forms of DON for growth, including several amino acids, purines, and pyrimidines. A cell‐surface protein up‐regulated in the absence of inorganic nitrogen, NRP1, is hypothesized to play a role in the metabolism of one or more of these organic nitrogen forms. Here, the genomic and cDNA sequence of NRP1 is reported. Structural predictions based on the amino acid sequence suggest a pyridoxal‐5′‐phosphate‐dependent enzyme that may have a role in acquiring nitrogen from amino acids. Further evidence for the function of NRP1 is measured in spent media from nitrogen‐limited cultures, which contain NRP1 and have glutaminase and formamidase activity. Field studies using an antibody to NRP1 show that it is expressed in E. huxleyi during bloom conditions in a Norwegian fjord.     

REVERSIBLE KINETIC MODEL FOR THE SHORT-TERM REGULATION OF METHYLAMMONIUM UPTAKE IN TWO PHYTOPLANKTON SPECIES,DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE) AND PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Methylammonium, an ammonium analog, was used to study the short-term kinetics of ammonium uptake in a diatom, Phaeodactylum tricornutum Bohlin, and a green alga, Dunaliella tertiolecta Butcher. Time courses of methylammonium disappearance were measured over a wide range of initial substrate concentrations for the two species. It was shown that feedback inhibition, described mathematically by a reversible enzyme kinetic model, can be used to explain the data. For the two species, there was good agreement between the kinetic parameters obtained from the analysis of the uptake versus substrate curve and those from the fit of the reversible kinetic model to the time-course data. All time courses of CH₃NH₃⁺ disappearance could be described by constants V_m and k_s. Ammonium time-course data show some similarities to its analog, methylammonium. Our study suggests that the apparent change in V_m and k_s with time measured after the addition of saturating ammonium concentrations reflects an uncoupling between transport and assimilation of the substrate rather than a real change in the kinetic parameters of the transport mechanism.     

EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE): NITROGEN‐METABOLISM GENES AND THEIR EXPRESSION IN RESPONSE TO EXTERNAL NITROGEN SOURCES1

The availability and composition of dissolved nitrogen in ocean waters are factors that influence species composition in natural phytoplankton communities. The same factors affect the ratio of organic to inorganic carbon incorporation in calcifying species, such as the coccolithophore Emiliania huxleyi (Lohman) W. W. Hay et H. Mohler. E. huxleyi has been shown to thrive on various nitrogen sources, including dissolved organic nitrogen. Nevertheless, assimilation of dissolved nitrogen under nitrogen‐replete and ‐limited conditions is not well understood in this ecologically important species. In this study, the complete amino acid sequences for three functional genes involved in nitrogen metabolism in E. huxleyi were identified: a putative formamidase, a glutamine synthetase (GSII family), and assimilatory nitrate reductase. Expression patterns of the three enzymes in cells grown on inorganic as well as organic nitrogen sources indicated reduced expression levels of nitrate reductase when cells were grown on NH₄⁺ and a reduced expression level of the putative formamidase when growth was on NO₃^?. The data reported here suggest the presence of a nitrogen preference hierarchy in E. huxleyi. In addition, the gene encoding for a phosphate repressible phosphate permease was more highly expressed in cells growing on formamide than in cells growing on inorganic nitrogen sources. This finding suggests a coupling between phosphate and nitrogen metabolism, which might give this species a competitive advantage in nutrient‐depleted environments. The potential of using expression of genes investigated here as indicators of specific nitrogen‐metabolism strategies of E. huxleyi in natural populations of phytoplankton is discussed.     

GROWTH AND PHOTOSYNTHESIS OF ULVA ROTUNDATA (CHLOROPHYTA) AS A FUNCTION OF TEMPERATURE AND SQUARE WAVE IRRADIANCE IN INDOOR CULTURE1

Temperature and photon flux density (PFD) vary independently in estuaries, e.g. high PFD may occur at any temperature, so it is necessary to consider synergistic effects of these factors on algal growth. Because natural PFD is highly variable and daylength changes confound seasonal temperature cycles, it is easier to interpret factorial experiments in controlled laboratory conditions. Clonal Ulva rotundata Blid. (Chlorophyta) has been studied extensively in outdoor culture. In this study it was maintained indoors under square wave photoperiods at five PFDs and three temperatures. Growth rate, photqsynthetic light response (P-I) curves, and photosystem II chlorophyll fluorescence properties were measured at the growth temperature following acclimation. Interactions between PFD and growth temperature were strongly indicated in all physiological parameters measured. Greatest PFD response occurred at the highest temperature, and the largest temperature response occurred at the highest PFD. Light-saturated photosynthesis (P_m) dark respiration (R_d), and light-limited quantum yield (Φ_m) were sufficient to describe acclimation status. The light-saturation parameter (I_k) was redundant and potentially misleading. Although U. rotundata exhibits a great amplitude of photoacclimation, it apparently has little capacity for temperature acclimation compared to the kelp, Laminaria saccharina, for which published data indicate similar photosynthetic rates over a broad range of growth temperatures. Diurnal variation of P_m and R_d at a growth PFD of ～ 1700 ± 200 μmol photons · m^?2· s^?1 was similar to the pattern observed previously in outdoor culture, suggesting endogenous control of these parameters. Quantum yield and the ratio of variable to maximum chlorophyll fluorescence (F_v/F_m), which were depressed in midday sunlight exceeding ～ 1500 μmol photons · m^?2· s^?1, were relatively invariant through the day in indoor culture, indicating that these parameters are controlled primarily by instantaneous PFD. Growth and fluorescence data are also presented for some other macroalgae for comparative purposes.