INFLUENCE OF LOW LIGHT AND A LIGHT: DARK CYCLE ON NO3– UPTAKE,INTRACELLULAR NO3–, AND NITROGEN ISOTOPE FRACTIONATION BY MARINE PHYTOPLANKTON1

The nitrogen isotope enrichment factor (ɛ) of four species of marine phytoplankton grown in batch cultures was determined during growth in continuous saturating light, continuous low light, and a 12:12‐h light:dark cycle, with nitrate as a nitrogen source. The low growth rate that resulted from low irradiance caused an increased accumulation of the intracellular nitrate pool and/or a reduction in cell volume and was correlated to a species‐specific increase in the measured ɛ value, compared with the saturating light conditions. The largest response was in the diatom Thalassiosira weissflogii (Grun.) Fryxell et Hasle, which showed a nearly 3‐fold increase between high and low light conditions (6.2–15.2‰). The smallest response was in T. pseudonana (Hustedt) Hasle et Heimdal, which showed no change in the ɛ value of approximately 5‰ in both high and low light conditions. There was significant but smaller increases in the ɛ value for the diatom T. rotula Meunier (2.7–5.6‰) and the prymnesiophyte Emiliania huxleyi (Lohm.) Hay et Mohler (4.5–9.4‰) between high and low light levels. In the light:dark experiments, all three diatoms but not the prymnesiophyte exhibited an increase in ɛ. This increase was linked to the ability of diatoms to assimilate nitrate at night. The results of the these experiments suggest that the light regime influences the relative uptake, assimilation, and efflux rates of nitrate and results in differences in the expression of the isotope effect by the enzyme nitrate reductase. Therefore, variations in nitrate isotope fractionation in nature can be more accurately interpreted when the light regime and species composition are taken into consideration.     

Intron features of key functional genes mediating nitrogen metabolism in marine phytoplankton

Introns are widespread and variable in eukaryotic genomes. Although their histories and functions, or even whether all of them have any function, remain largely unknown, analysis of intron sequences and genomic contexts may shed light on the evolutionary history of genes and organisms. The number and frequency of introns vary widely in the small number of published genomes of protists and algae suggesting that the same is true of the vast diversity of protists and algae that remain uncultivated. The objective of this study were to investigate introns in sequences of functional genes of phytoplankton, both in published genomes and in sequences obtained from environmental clone libraries. We examined the introns of the genes involved in nitrogen uptake and assimilation pathways in the genome sequences of cultivated phytoplankton as well as in environmental clone libraries of nitrate reductases (NR), nitrite reductase (NiR), nitrate transporter (Nrt2) and ammonium transporter (AMT) genes constructed from pelagic phytoplankton communities in Monterey Bay (CA, USA) and Onslow Bay (NC, USA). Here we describe the most extensive set to date of intron sequences from uncultivated marine algae and report important differences for diatom vs. non-diatom sequences. The majority of the introns in NR, NiR, Nrt2 and AMT from cultured phytoplankton and environmental libraries showed canonical splice patterns. Introns found in diatom-like NR environmental libraries had lower GC content than the respective exons. The green algal-like NR and Nrt2 environmental sequences had introns and exons of much more similar GC content, and both higher than in diatoms. These patterns suggest a different evolutionary history and recent acquisition of diatom introns compared to other algae.     

GROWTH INHIBITION AND TOXICITY OF THE DIATOM ALDEHYDE 2‐TRANS, 4‐TRANS‐DECADIENAL ON THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)1

A common aldehyde present in marine and freshwater diatoms, 2‐trans, 4‐trans‐decadienal (A3), is involved in the wound‐activated response of diatoms to copepod grazing. Upon breakage of the diatom cell membrane, aldehydes are enzymatically produced by the rapid conversion of precursors and strongly impact copepod reproduction by impairing egg production and hatching success, inducing teratogenic embryos modifications. In this study, A3 was assayed with the marine diatom Thalassiosira weissflogii (Grunow) Fryxell et Hasle. The aldehyde concentration necessary to reduce 50% growth rate (EC₅₀) was 0.29 mg·L^?1. Decadienal was found to inhibit T. weissflogii cell growth in a dose‐ and time‐dependent manner, with irreversible effects after 24 h of exposure. Decadienal induced a degenerative process, through modifications of cell membrane characteristics, interference with cell cycle progression, and with cell metabolic activity, leading to cell death. A preferential action of A3 on dividing cells was observed. Photosynthetic efficiency significantly decreased upon exposure to the aldehyde, paralleled by an increase in diatoxanthin, suggesting a protective role of this xanthophyll, usually involved in photoprotection. Dying cells exhibited the morphological and biochemical features that bear close resemblance to apoptosis of mammalian cells, including cell shrinkage, chromatin condensation, and degradation of nuclear DNA to nucleosomal size fragments. These data are the first direct evidence to show aldehydes are toxic to diatoms. We suggest a possible nontoxic role of such compounds as chemical signals of unfavorable conditions within the phytoplankton communities, which may be relevant for the population dynamics of diatoms during blooms.     

Comparison of population growth and photosynthetic apparatus changes in response to different nutrient status in a diatom and a coccolithophore

In many marine ecosystems, diatoms dominate in nutrient‐rich coastal waters while coccolithiophores are found offshore in areas where nutrients may be limiting. In lab‐controlled batch cultures, mixed‐species competition between the diatom Phaeodactylum tricornutum and the coccolithophore Emiliana huxleyi and the response of each species were examined under nitrate (N) and phosphate (P) starvation. Based on the logistic growth model and the Lotka–Volterra competition model, E. huxleyi showed higher competitive abilities than P. tricornutum under N and P starvation. For both species, cell growth was more inhibited by P starvation, while photosynthetic functions (chl a fluorescence parameters) and cellular constituents (pigments) were impaired by N starvation. The decline of photosynthetic functions occurred later in E. huxleyi (day 12) than in P. tricornutum (day 9); this time difference was associated with greater damage of the photosynthetic apparatus in P. tricornutum compared with E. huxleyi. Xanthophyll cycle pigment accumulation and the transformation from diadinoxanthin to diatoxanthin was more active in E. huxleyi than P. tricornutum, under similar N and P starvation. We concluded that E. huxleyi and P. tricornutum have different mechanisms to allocate resources and energy under nutrient starvation. It appears that E. huxleyi has a more economic strategy to adapt to nutrient depleted environments than P. tricornutum. These findings provided additional evidence explaining how N versus P limitation differentially support diatom and coccolithophore blooms in natural environments.     

The physiological response of marine diatoms to ocean acidification: differential roles of seawater pCO2 and pH

Although increasing the pCO₂ for diatoms will presumably down‐regulate the CO₂‐concentrating mechanism (CCM) to save energy for growth, different species have been reported to respond differently to ocean acidification (OA). To better understand their growth responses to OA, we acclimated the diatoms Thalassiosira pseudonana, Phaeodactylum tricornutum, and Chaetoceros muelleri to ambient (pCO₂ 400 μatm, pH 8.1), carbonated (pCO₂ 800 μatm, pH 8.1), acidified (pCO₂ 400 μatm, pH 7.8), and OA (pCO₂ 800 μatm, pH 7.8) conditions and investigated how seawater pCO₂ and pH affect their CCMs, photosynthesis, and respiration both individually and jointly. In all three diatoms, carbonation down‐regulated the CCMs, while acidification increased both the photosynthetic carbon fixation rate and the fraction of CO₂ as the inorganic carbon source. The positive OA effect on photosynthetic carbon fixation was more pronounced in C. muelleri, which had a relatively lower photosynthetic affinity for CO₂, than in either T. pseudonana or P. tricornutum. In response to OA, T. pseudonana increased respiration for active disposal of H⁺ to maintain its intracellular pH, whereas P. tricornutum and C. muelleri retained their respiration rate but lowered the intracellular pH to maintain the cross‐membrane electrochemical gradient for H⁺ efflux. As the net result of changes in photosynthesis and respiration, growth enhancement to OA of the three diatoms followed the order of C. muelleri > P. tricornutum > T. pseudonana. This study demonstrates that elucidating the separate and joint impacts of increased pCO₂ and decreased pH aids the mechanistic understanding of OA effects on diatoms in the future, acidified oceans.     

Effects of non-steady-state iron limitation on nitrogen assimilatory enzymes in the marine diatom thalassiosira weissflogii (BACILLARIOPHYCEAE)

Since the recognition of iron‐limited high nitrate (or nutrient) low chlorophyll (HNLC) regions of the ocean, low iron availability has been hypothesized to limit the assimilation of nitrate by diatoms. To determine the influence of non‐steady‐state iron availability on nitrogen assimilatory enzymes, cultures of Thalassiosira weissflogii (Grunow) Fryxell et Hasle were grown under iron‐limited and iron‐replete conditions using artificial seawater medium. Iron‐limited cultures suffered from decreased efficiency of PSII as indicated by the DCMU‐induced variable fluorescence signal (F_v/F_m). Under iron‐replete conditions, in vitro nitrate reductase (NR) activity was rate limiting to nitrogen assimilation and in vitro nitrite reductase (NiR) activity was 50‐fold higher. Under iron limitation, cultures excreted up to 100 fmol NO₂^?·cell^?1·d^?1 (about 10% of incorporated N) and NiR activities declined by 50‐fold while internal NO₂^? pools remained relatively constant. Activities of both NR and NiR remained in excess of nitrogen incorporation rates throughout iron‐limited growth. One possible explanation is that the supply of photosynthetically derived reductant to NiR may be responsible for the limitation of nitrogen assimilation at the NO₂^? reduction step. Urease activity showed no response to iron limitation. Carbon:nitrogen ratios were equivalent in both iron conditions, indicating that, relative to carbon, nitrogen was assimilated at similar rates whether iron was limiting growth or not. We hypothesize that, diatoms in HNLC regions are not deficient in their ability to assimilate nitrate when they are iron limited. Rather, it appears that diatoms are limited in their ability to process photons within the photosynthetic electron transport chain which results in nitrite reduction becoming the rate‐limiting step in nitrogenassimilation.     

Increased intrusion of warming Atlantic water leads to rapid expansion of temperate phytoplankton in the Arctic

The Arctic Ocean and its surrounding shelf seas are warming much faster than the global average, which potentially opens up new distribution areas for temperate‐origin marine phytoplankton. Using over three decades of continuous satellite observations, we show that increased inflow and temperature of Atlantic waters in the Barents Sea resulted in a striking poleward shift in the distribution of blooms of Emiliania huxleyi, a marine calcifying phytoplankton species. This species' blooms are typically associated with temperate waters and have expanded north to 76°N, five degrees further north of its first bloom occurrence in 1989. E. huxleyi's blooms keep pace with the changing climate of the Barents Sea, namely ocean warming and shifts in the position of the Polar Front, resulting in an exceptionally rapid range shift compared to what is generally detected in the marine realm. We propose that as the Eurasian Basin of the Arctic Ocean further atlantifies and ocean temperatures continue to rise, E. huxleyi and other temperate‐origin phytoplankton could well become resident bloom formers in the Arctic Ocean.     

Effects of light and nutrients on seasonal phytoplankton succession in a temperate eutrophic coastal lagoon

Rodeo Lagoon, a low-salinity coastal lagoon in the Golden Gate National Recreation Area, California, United States, has been identified as an important ecosystem due to the presence of the endangered goby (Eucyclogobius newberri). Despite low anthropogenic impacts, the lagoon exhibits eutrophic conditions and supports annual episodes of very high phytoplankton biomass. Weekly assessments (February–December 2007) of phytoplankton indicated diatoms, Nodularia spumigena, Chaetoceros muelleri var. muelleri, flagellated protozoa, a mixed assemblage, and Microcystis aeruginosa dominated the algal community in successive waves. Phytoplankton succession was significantly correlated (r ² = 0.37, p < 0.001) with averaged daily irradiance (max = 29.7 kW m⁻² d⁻¹), water column light attenuation (max = 14 m⁻¹), and orthophosphate and dissolved inorganic carbon concentrations (max = 1.5 and 2920 μM, respectively). Negative effects of phytoplankton growth and decay included excessive ammonia concentrations (exceeded EPA guidelines on 77% of sampling days), hypoxia (<3 mg l⁻¹ dissolved oxygen), and introduction of several microcystins, all in the latter half of the year. Our one-year study suggests that this coastal lagoon is a highly seasonal system with strong feedbacks between phytoplankton and geochemical processes.     

Influence of sea ice on the composition of the spring phytoplankton bloom in the northern Baltic Sea

During the late winter and spring of 1994, the influence of sea ice on phytoplankton succession in the water was studied at a coastal station in the northern Baltic Sea. Ice cores were taken together with water samples from the underlying water and analysed for algal composition, chlorophyll a and nutrients. Sediment traps were placed under the ice and near the bottom, and the sedimented material was analysed for algal composition. The highest concentration of ice algae (4.1 mmol C m⁻²) was found shortly before ice break-up in the middle of April, coincidental with the onset of an under-ice phytoplankton bloom. The ice algae were dominated by the diatoms Chaetoceros wighamii Brightwell, Melosira arctica (Ehrenberg) Dickie and Nitzschia frigida Grunow. Under the ice the diatom Achnanthes taeniata Grunow and the dinoflagellate Peridiniella catenata (Levander) Balech were dominant. Calculations of sinking rates and residence times of the dominant ice algal species in the photic water column indicated that only one ice algal species (Chaetoceros wighamii) had a seeding effect on the water column: this diatom dominated the spring phytoplankton bloom in the water together with Achnanthes taeniata and Peridiniella catenata. Received: 9 May 1997 / Accepted: 15 February 1998     

Detection of a variable intracellular acid‐labile carbon pool in Thalassiosira weissflogii (Heterokontophyta) and Emiliania huxleyi (Haptophyta) in response to changes in the seawater carbon system

Accumulation of an intracellular pool of carbon (C_i pool) is one strategy by which marine algae overcome the low abundance of dissolved CO₂ (CO_2(aq)) in modern seawater. To identify the environmental conditions under which algae accumulate an acid‐labile C_i pool, we applied a ¹⁴C pulse‐chase method, used originally in dinoflagellates, to two new classes of algae, coccolithophorids and diatoms. This method measures the carbon accumulation inside the cells without altering the medium carbon chemistry or culture cell density. We found that the diatom Thalassiosira weissflogii [(Grunow) G. Fryxell & Hasle] and a calcifying strain of the coccolithophorid Emiliania huxleyi [(Lohmann) W. W. Hay & H. P. Mohler] develop significant acid‐labile C_i pools. Ci pools are measureable in cells cultured in media with 2–30 µmol l^?1 CO_2(aq), corresponding to a medium pH of 8.6–7.9. The absolute C_i pool was greater for the larger celled diatoms. For both algal classes, the C_i pool became a negligible contributor to photosynthesis once CO_2(aq) exceeded 30 µmol l^?1. Combining the ¹⁴C pulse‐chase method and ¹⁴C disequilibrium method enabled us to assess whether E. huxleyi and T. weissflogii exhibited thresholds for foregoing accumulation of DIC or reduced the reliance on bicarbonate uptake with increasing CO_2(aq). We showed that the C_i pool decreases with higher CO₂:HCO₃^? uptake rates.     

Energy cost and putative benefits of cellular mechanisms modulating buoyancy in aflagellate marine phytoplankton

Little information is available on the energetics of buoyancy modulation in aflagellate phytoplankton, which comprises the majority of autotrophic cells found in the ocean. Here, we computed for three aflagellate species of marine phytoplankton (Emiliania huxleyi, Thalassiosira pseudonana, and Ethmodiscus rex) the theoretical minimum energy cost as photons absorbed and nitrogen resource required of the key physiological mechanisms (i.e., replacement of quaternary ammonium by dimethyl‐sulfoniopropionate, storage of polysaccharides, and cell wall biosynthesis) affecting the cell's vertical movement as a function of nitrogen (N) availability. These energy costs were also normalized to the capacity of each buoyancy mechanism to modulate sinking or rising rates based on Stokes' law. The three physiological mechanisms could act as ballast in the three species tested in conditions of low N availability at a low fraction (<12%) of the total photon energy cost for growth. Cell wall formation in E. huxleyi was the least costly ballast strategy, whereas in T. pseudonana, the photon energy cost of the three ballast strategies was similar. In E. rex, carbohydrate storage and mobilization appear to be energetically cheaper than modulations in organic solute synthesis to achieve vertical migration. This supports the carbohydrate‐ballast strategy for vertical migration for this species, but argues against the theory of replacement of low‐ or high‐density organic solutes. This study brings new insights into the energy cost and potential selective advantages of several strategies modulating the buoyancy of aflagellate marine phytoplankton.     

The 24 hour recovery kinetics from n starvation in Phaeodactylum tricornutum and Emiliania huxleyi

The response of N (nitrate) starved cells of the diatom Phaeodactylum tricornutum and the coccolithophore Emiliania huxleyi to a pulse of new N were measured to investigate rapid cellular and photosynthetic recovery kinetics. The changes of multiple parameters were followed over 24 h. In P. tricornutum, the recovery of F_v/F_m (the maximum quantum yield of PS II) and σ_PSII (the functional absorption cross‐section for PSII) started within the first hour, much earlier than other parameters. Cellular pigments did not recover during the 24 h but the chlorophyll (chl) a/carotenoid ratios increased to levels measured in the controls. Cell division was independent of the recovery of chl a. In E. huxleyi, the recovery of F_v/F_m and σ_PSII started after an hour, synchronous with the increase in cellular organic N and chl a with pigments fully recovered within 14 h. P. tricornutum prioritized the recovery of its photosynthetic functions and cell divisions while E. huxleyi did not follow this pattern. We hypothesize that the different recovery strategies between the two species allow P. tricornutum to be more competitive when N pulses are introduced into N‐limited water while E. huxleyi is adapted to N scarce waters where such pulses are infrequent. These findings are consistent with successional patterns observed in coastal environments. This is one of only a few studies exploring recovery kinetics of cellular functions and photosynthesis after nitrogen stress in phytoplankton. Our results can be used to enhance ecological models linking phytoplankton traits to species diversity and community structure.     

PHYTOPLANKTON SELENIUM REQUIREMENTS: THE CASE FOR SPECIES ISOLATED FROM TEMPERATE AND POLAR REGIONS OF THE SOUTHERN HEMISPHERE1

A series of laboratory culture experiments was used to investigate the effect of selenium (Se, 0–10 nM) on the growth, cellular volume, photophysiology, and pigments of two temperate and four polar oceanic phytoplankton species [coccolithophore Emiliania huxleyi (Lohmann) W. W. Hay et H. P. Mohler, cyanobacterium Synechococcus sp., prymnesiophyte Phaeocystis sp., and three diatoms—Fragilariopsis cylindrus (Grunow) Kriegar, Chaetoceros sp., and Thalassiosira antarctica G. Karst.]. Only Synechoccocus sp. and Phaeocystis sp. did not show any requirement for Se. Under Se‐deficient conditions, the growth rate of E. huxleyi was decreased by 1.6‐fold, whereas cellular volume was increased by 1.9‐fold. Se limitation also decreased chl a (2.5‐fold), maximum relative electron transport rate (1.9‐fold), and saturating light intensity (2.8‐fold), suggesting that Se plays a role in photosynthesis or high‐light acclimation. Pigment analysis for Antarctic taxa provided an interesting counterpoint to the physiology of E. huxleyi. For all Se‐dependent Antarctic diatoms, Se limitation decreased growth rate and chl a content, whereas cellular volume was not affected. Pigment analysis revealed that other pigments were affected under Se deficiency. Photoprotective pigments increased by 1.4‐fold, while diadinoxanthin:diatoxanthin ratios decreased by 1.5‐ to 4.9‐fold under Se limitation, supporting a role for Se in photoprotection. Our results demonstrate an Se growth requirement for polar diatoms and indicate that Se could play a role in the biogeochemical cycles of other nutrients, such as silicic acid in the Southern Ocean. Se measurements made during the austral summer in the Southern Ocean and Se biological requirement were used to discuss possible Se limitation in phytoplankton from contrasting oceanographic regions.     

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.     

INTER‐ AND INTRASPECIFIC RELATIONSHIPS BETWEEN NUCLEAR DNA CONTENT AND CELL SIZE IN SELECTED MEMBERS OF THE CENTRIC DIATOM GENUS THALASSIOSIRA (BACILLARIOPHYCEAE)1

The enormous species diversity of diatoms correlates with the remarkable range of cell sizes in this group. Nuclear DNA content relates fundamentally to cell volume in other eukaryotic cells. The relationship of cell volume to G1 DNA content was determined among selected members of the genus Thalassiosira, one of the most species‐rich and well‐studied centric diatom genera. Both minimum and maximum species‐specific cell volume correlated positively with G1 DNA content. Phylogeny based on 5.8 S and ITS rDNA sequences indicated that multiple changes in G1 DNA content and cell volume occurred in Thalassiosira evolution, leading to a 1,000‐fold range in both parameters in the group. Within the Thalassiosira weissflogii (Grunow) G. A. Fryxell et Grunow species complex, G1 DNA content varied 3‐fold: differences related to geographic origin and time since isolation; doubling and tripling of G1 DNA content occurred since isolation in certain T. weissflogii isolates; and subcultures of T. weissflogii CCMP 1336 diverged in DNA content by 50% within 7 years of separation. Actin, β‐tubulin, and Spo11/TopVIA genes were selected for quantitative PCR estimation of haploid genome size in subclones of selected T. weissflogii isolates because they occur only once in the T. pseudonana Hasle et Heimdal genome. Comparison of haploid genome size estimates with G1 DNA content suggested that the most recent T. weissflogii isolate was diploid, whereas other T. weissflogii isolates appeared to be polyploid and/or aneuploid. Aberrant meiotic and mitotic cell divisions were observed, which might relate to polyploidization. The structural flexibility of diatom genomes has important implications for their evolutionary diversification and stability during laboratory maintenance.