Chlorophyte Dunaliella Tertiolecta Research Articles

Novel incubation‐free approaches to determine phytoplankton net primary productivity, growth, and biomass based on flow cytometry and quantification of ATP and NAD(H)

AbstractRecent advances in sorting flow cytometry (SFCM) and sensitive biochemical techniques have opened the door for a variety of new incubation‐free measurements to assess cell physiology. We adapted highly sensitive bioluminescent methodologies to measure cell NAD(H) and adenosine triphosphate (ATP) contents as predictors of phytoplankton primary production and growth rate. We first applied these methods to algae in taxonomically distinct groups: the chlorophyte Dunaliella tertiolecta, the diatom Thalassiosira pseudonana and a model cyanobacterium Synechococcus sp. WH8102. We found that NAD(H) content [NAD(H)phyto] increased significantly with increasing net primary productivity (NPP) and growth rate. In contrast, and in agreement with previous studies, phytoplankton ATP content (ATPphyto) was highly correlated with carbon biomass. Furthermore, ATPphyto was not correlated with NPP or growth rate. As a field test of the method, we then measured NAD(H)phyto in phytoplankton samples collected by SFCM in the North Pacific. These measurements yielded an estimate of NPP closely matching that from the traditional incubation‐dependent 14C uptake technique. The methods described here enable low‐biomass, incubation‐free estimates of phytoplankton NPP, growth rate, and biomass.

Low-steady-state metabolism induced by elevated CO2 increases resilience to UV radiation in the unicellular green-algae Dunaliella tertiolecta

Global change factors derived from anthropogenic activities such as increased CO2 and ultraviolet radiation (UVR) have direct impacts on the physiological responses of organisms in both terrestrial and aquatic ecosystems. We assessed in this work the mechanistic, physiological and molecular response of the unicellular chlorophyte Dunaliella tertiolecta exposed to combinatorial present (390 ppmv, LC) and future CO2 levels predicted for the year 2100 (900 ppmv, HC) and different UVR doses. Growth rates, cell esterase activity, reactive oxygen species accumulation (ROS), and 14C fixation decreased under HC compared to LC. The deleterious effects of UVR were attenuated in HC, except for decreased photosynthetic capacities. Transcriptome analysis showed different expression patterns depending on UVR and CO2 levels. PsbA, LhcII and eCA gene expression was downregulated at HC compared to LC. However, photolyase (PL) gene expression was upregulated at HC with respect LC in UVR. This suggests that HC (as opposed to LC) promoted a “low-metabolic steady state”, decreasing UVR exposure-activated repair by means of protein replacement. Only PL photoreactivation was active under HC. Our data add evidence on D. tertiolecta possibly becoming more resilient to UVR exposure under future CO2 regimes, and maybe other marine unicellular chlorophytes, warranting serious considerations on the consequent ecological implications.

Effect on Growth, Photosynthesis, and Oxidative Stress of Single Walled Carbon Nanotubes Exposure to Marine Alga Dunaliella tertiolecta.

Single walled carbon nanotubes were carboxylated by microwave assisted acid oxidation (f-SWCNTs) and examined for their ecotoxicity on marine alga chlorophyte Dunaliella tertiolecta. Toxicity was evaluated based on growth, photosynthetic activities, oxidative stress, and intracellular glutathione in the concentration range of 0.1-20 mg/L f-SWCNT. Physical interactions between the f-SWCNT and alga were examined using light microscopy and scanning electron microscope. Increasing the nanotube concentration increased the toxic effects where growth inhibition was as high as 30%, photosynthetic yield decreased by as much as 18%, and intracellular glutathione reduction reached 95%. The results from f-SWCNTs were somewhat different when compared to our previous study using the same algae and functionalized multiwalled carbon nanotubes, where exposure led to longer lag phase and higher growth rate inhibition.

Elevated CO2 alleviates high PAR and UV stress in the unicellular chlorophyte Dunaliella tertiolecta.

The effects of increased CO2 and irradiance on the physiological performance of the chlorophyte Dunaliella tertiolecta were studied at different PAR and UVR (UVA + UVB) irradiances, simulating the solar radiation at different depths, at present (390 ppmv, LC) and predicted CO2 levels for the year 2100 (1000 ppmv, HC). Elevated CO2 resulted in higher optimum and effective quantum yields (F(v)/F(m) and ϕPSII, respectively), electron transport rates (ETR) and specific growth rates (μ). Cell stress was alleviated in HC with respect to LC as evidenced by a decrease in reactive oxygen species (ROS) accumulation. DNA damage showed a 42-fold increase in cyclobutane-pyrimidine dimer (CPD) formation under the highest irradiance (1100 μmol quanta m(-2) s(-1)) in LC with respect to the lowest irradiance (200 μmol quanta m(-2) s(-1)). Photolyase (CII-PCD-PL) gene expression was upregulated under HC resulting in a drastic decrease in CPD accumulation to only 25% with respect to LC. Proliferating cell nuclear antigen (PCNA) accumulation was always higher in HC and the accumulation pattern indicated its involvement in repair or growth depending on the irradiance dose. The repressor of silencing (ROS1) was only marginally involved in the response, suggesting that photoreactivation was the most relevant mechanism to overcome UVR damage. Our results demonstrate that future scenarios of global change result in alleviation of irradiance stress by CO2-induced photoprotection in D. tertiolecta.

Mono- and digalactosyldiacylglycerol composition of dinoflagellates. VII. Evidence against galactolipid production and plastid presence in the heterotrophic, basal dinoflagellate, Oxyrrhis marina

The heterotrophic genus Oxyrrhis contains two species, O. marina and O. maritima, which occupy positions basal to the dinoflagellate lineage. Oxyrrhis is also related to apicomplexan parasites, which have recently been shown to have a non-photosynthetic, relic plastid referred to as the apicoplast. A recent study by Slamovits & Keeling (2008) demonstrated the presence of plastid-targeted proteins within O. marina. We hypothesized that if O. marina does indeed have plastids, then mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively), which are the two most prominent plastidial membrane lipids, would be present. Therefore, we examined three isolates of O. marina to determine if they can produce MGDG and DGDG. We observed that O. marina, when fed the chlorophyte Dunaliella tertiolecta, possessed forms of MGDG and DGDG containing a C18:3 fatty acid at the sn-1 position and most containing a C16:3-4 fatty acid at the sn-2 position; these were derived solely from the prey itself. Examination of published expressed sequence tag (EST) and transcriptome databases of O. marina for the genes encoding MGDG and DGDG synthases, two enzymes integral to the incorporation of galactose in the final forms of these lipids, failed to reveal their presence. Taken together, these results indicate that O. marina does not produce a non-photosynthetic, relictual plastid. However, the presence of plastid-targeted proteins may indicate that O. marina maintains, however briefly, plastids acquired from its prey in a form of kleptoplasty that has been observed previously in dinoflagellates.

Toxic effects of ZnO nanoparticles towards marine algae Dunaliella tertiolecta

Dose response curve and population growth rate alterations of marine Chlorophyte Dunaliella tertiolecta derived from the exposure to ZnO nanoparticles were evaluated. Bulk ZnO and ionic zinc were also investigated for comparison. At the same time, the aggregation state and particle size distribution were monitored. The evaluated 50% effect concentration (EC50 1.94 [0.78–2.31]mgZnL−1) indicates that nano ZnO is more toxic than its bulk counterpart (EC50 3.57 [2.77–4.80]mgZnL−1). Cross-referencing the toxicity parameters calculated for ZnCl2 (EC50 0.65 [0.36–0.70]mgZnL−1) and the dissolution properties of the ZnO, it can be gathered that the higher toxicity of nano ZnO is most likely related to the peculiar physicochemical properties of the nanostate with respect to the bulk material. Furthermore growth rate of D. tertiolecta was significantly affected by nano ZnO exposure.Our findings suggest that the primary particle size of the dispersed particles affect the overall toxicity.

Comparative responses of two species of marine phytoplankton to metolachlor exposure

Metolachlor, a chloroacetanilide herbicide, has been frequently detected in coastal waters. This study examined the growth, photosynthesis, and detoxification responses of chlorophyte Dunaliella tertiolecta (DT) and brown tide alga Aureococcus anophagefferens (AA) upon 5-day exposure to 0.5–5mgL−1 metolachlor. Growth was assessed with exponential growth rate, and 5th day in vivo chlorophyll fluorescence, chlorophyll a, b or c, cell density and cell size. The photosynthesis function was assessed with photochemical parameters of photosystem II (PSII) during the mid-exponential growth phase (i.e. 2–4 day metolachlor exposure). The biochemical detoxification was analyzed with glutathione production and metolachlor degradation. Results show that metolachlor caused up to ∼9% inhibition in growth rate in both species and an expected ∼35% and 25% inhibition in chlorophyll based endpoints in DT and AA respectively. DT had an up to 70% inhibition in cell density, but AA a 35% hormesis at 1mgL−1 metolachlor and no significant inhibition, as compared to the controls. Both DT and AA's cell sizes were enlarged by metolachlor exposure, but greater in DT (1.2% per mgL−1) than in AA (0.68% per mgL−1). On PSII photochemistry, maximum quantum yield was not affected in both species; PSII optical cross section and connectivity factor increased in DT but decreased in AA, suggesting species specific impact on PSII function. On detoxification responses, glutathione production, when normalized to total chlorophyll a, was not affected by metolachlor in both species; further, despite of heterotrophic capacity of A. anophagefferens metolachlor was not significantly degraded by this alga during the 5-day incubation. The species specific effects on algal growth have ecological implications of potential selective inhibition of chlorophytes by metolachlor herbicide.

Cell survival after UV radiation stress in the unicellular chlorophyte Dunaliella tertiolecta is mediated by DNA repair and MAPK phosphorylation

Ultraviolet radiation (UVR) induces damage in a variety of organisms, and cells may adapt by developing repair or tolerance mechanisms to counteract such damage; otherwise, the cellular fate is cell death. Here, the effect of UVR-induced cell damage and the associated signalling and repair mechanisms by which cells are able to survive was studied in Dunaliella tertiolecta. UVR did not cause cell death, as shown by the absence of SYTOX Green-positive labelling cells. Ultrastructure analysis by transmission electron microscopy demonstrated that the cells were alive but were subjected to morphological changes such as starch accumulation, chromatin disaggregation, and chloroplast degradation. This behaviour paralleled a decrease in F v/F m and the formation of cyclobutane–pyrimidine dimers, showing a 10-fold increase at the end of the time course. There was a high accumulation of the repressor of transcriptional gene silencing (ROS1), as well as the cell proliferation nuclear antigen (PCNA) in UVR-treated cells, revealing activation of DNA repair mechanisms. The degree of phosphorylation of c-Jun N-terminal kinase (JNK) and p38-like mitogen-activated protein kinases was higher in UVR-exposed cells; however, the opposite occurred with the phosphorylated extracellular signal-regulated kinase (ERK). This confirmed that both JNK and p38 need to be phosphorylated to trigger the stress response, as well as the fact that cell division is arrested when an ERK is dephosphorylated. In parallel, both DEVDase and WEHDase caspase-like enzymatic activities were active even though the cells were not dead, suggesting that these proteases must be considered within a wider frame of stress proteins, rather than specifically being involved in cell death in these organisms.

Functional response of carbon absorption efficiency in the pelagic calanoid copepod Acartia tonsa

We constructed a general relationship between prey concentration and absorption efficiency (AE; previously termed assimilation efficiency) in the cosmopolitan calanoid copepod Acartia tonsa. The relationships between observed AEs and prey concentrations developed differently on different prey species. While AEs decreased asymptotically with prey concentration on the cryptophyte Rhodomonas baltica, the chlorophyte Dunaliella tertiolecta generated a v‐shaped response, whereas AEs remained constant on the diatom Thalassiosira weissflogii. We used these observations to develop a mechanistic gut model in which gut carbon concentrations depend on ingestion rate and either egestion rate or absorption rate. To investigate which rate was more important for AE, two different model modes were run: absorption was set to vary according to absorptive enzyme kinetics and egestion set to relate linearly to gut carbon concentration, or the contrary, egestion was set to vary according to enzyme kinetics and absorption set to relate linearly to gut concentration. This approach revealed that copepod carbon AE is controlled primarily by egestion rate kinetics and is independent of absorptive enzyme kinetics. Accordingly, we suggest a simplified regression model for the functional response of AE in pelagic copepods based solely on rates of ingestion and egestion.

STATE TRANSITIONS AND NONPHOTOCHEMICAL QUENCHING DURING A NUTRIENT-INDUCED FLUORESCENCE TRANSIENT IN PHOSPHORUS-STARVED DUNALIELLA TERTIOLECTA(1).

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 (Pi )-uptake and (2) energy-dependent quenching to allow the nutrient-stressed cell to avoid photodamage from excess light energy during nutrient uptake.

Growth inhibition of cultured marine phytoplankton by toxic algal-derived polyunsaturated aldehydes

Several marine diatoms produce polyunsaturated aldehydes (PUAs) that have been shown to be toxic to a wide variety of model organisms, from bacteria to invertebrates. However, very little information is available on their effect on phytoplankton. Here, we expand previous studies to six species of marine phytoplankton, belonging to different taxonomic groups that are well represented in marine plankton. The effect of three PUAs, 2 E,4 E-decadienal, 2 E,4 E-octadienal and 2 E,4 E-heptadienal, was assessed on growth, cell membrane permeability, flow cytometric properties and morphology. A concentration-dependent reduction in the growth rate was observed for all cultures exposed to PUAs with longer-chained aldehydes having stronger effects on growth than shorter-chained aldehydes. Clear differences were observed among the different species. The prymnesiophyte Isochrysis galbana was the most sensitive species to PUA exposure with a lower threshold for an observed effect triggered by mean concentrations of 0.10 μmol L −1 for 2 E,4 E-decadienal, 1.86 μmol L −1 for 2 E,4 E-octadienal and 3.06 μmol L −1 for 2 E,4 E-heptadienal, and a 50% growth inhibition (EC 50) with respect to the control at 0.99, 2.25 and 5.90 μmol L −1 for the three PUAs, respectively. Alternatively, the chlorophyte Tetraselmis suecica and the diatom Skeletonema marinoi (formerly S. costatum) were the most resistant species with 50% growth inhibition occurring at concentrations at least two to three times higher than I. galbana. In all species, the three PUAs caused changes in flow cytometric measures of cell size and cell granulosity and increased membrane permeability, assessed using the viability stain SYTOX Green. For example, after 48 h 51.6 ± 2.6% of I. galbana cells and 15.0 ± 1.8% of S. marinoi cells were not viable. Chromatin fragmentation was observed in the dinoflagellate Amphidinium carterae while clear DNA degradation was observed in the chlorophyte Dunaliella tertiolecta. Concentrations used are in a significant range for affecting growth and performance of phytoplankton living in close vicinity of PUA-producing algae. Thus, PUAs may act as allelochemicals by mediating interactions among planktonic organisms.

Effect of inhibitors of protein synthesis and DNA replication on the induction of proteolytic activities, caspase-like activities and cell death in the unicellular chlorophyte Dunaliella tertiolecta

When the chlorophyte alga Dunaliella tertiolecta is placed in darkness, a form of programmed cell death with many similarities to apoptosis (including the induction of caspase-like proteases) is induced. Many uncertainties about this process remain, two of which are whether it requires protein synthesis and whether there is potential viral involvement. In order to examine the relationship between the induction and/or activation of proteolytic activities and the cell death event, we used inhibitors of cytoplasmic protein synthesis (cycloheximide, CHX), organellar protein synthesis (chloramphenicol, CAP) and DNA synthesis (mitomycin C, MMC). Use of MMC also allowed us to examine whether temperate viruses were present in the D. tertiolecta isolate, since MMC treatment has been shown to induce lytic cycles. Addition of protein synthesis inhibitors (100 μM CHX or 1500 μM CAP applied singly or both inhibitors added together) did not prevent cell death from occurring when cultures were placed in the dark. There were no differences in caseinolytic activities visualized using zymograms, or in caspase 1, 3, 8 and 9 -like activities. Surprisingly, 100 μM MMC prevented the cell death event. Caseinolytic activities that appeared in darkness in controls did not appear in MMC-treated cultures, and caspase-like activities remained the same as in controls maintained in the light. The lack of effect of CHX and CAP suggests that the cell death programme we observe does not depend on protein synthesis, but rather on post-translational modification of pre-existing proteins. Results for MMC discount the involvement of temperate virus, but are difficult to interpret. MMC affects DNA synthesis and presumably transcription, but since inhibitors of translation did not prevent cell death, it is not clear why inhibiting transcription would. MMC affects cell cycle progression and cell division cycles, thus these processes may play as yet unexplained roles in mediating the cell death process observed.

Bioavailability of particle‐associated silver, cadmium, and zinc to the estuarine amphipod Leptocheirus plumulosus through dietary ingestion

We conducted experiments to determine effects of particle type on assimilatory metal bioavailability to Leptocheirus plumulosus, an infaunal, estuarine amphipod that is commonly used in sediment toxicity tests. The following particles were used to represent natural food items encountered by this surface‐deposit and suspension‐feeding amphipod: bacterial exopolymeric sediment coatings, polymeric coatings made from Spartina alterniflora extract, amorphous iron oxide coatings, the diatom Phaeodactylum tricornutum, the chlorophyte Dunaliella tertiolecta, processed estuarine sediment, and fresh estuarine sediment. Bioavailability of the gamma‐emitting radioisotopes 110mAg, 109Cd, and 65Zn was measured as the efficiency with which L. plumulosus assimilated metals from particles using pulse‐chase methods. Ag and Cd assimilation efficiencies were highest from bacterial exopolymeric coatings. Zn assimilation efficiency exhibited considerable interexperimental variation; the highest Zn assimilation efficiencies were measured from phytoplankton and processed sediment. In general, Ag and Cd assimilation efficiencies from phytoplankton were low and not related to the proportion of metal associated with cell cytosol or cytoplasm, a phenomenon reported for other particle‐ingesting invertebrates. Amphipod digestive processes explain differences in Ag and Cd assimilation efficiencies between exopolymeric coatings and phytoplankton. Results highlight the importance of labile polymeric organic carbon sediment coatings in dietary metals uptake by this benthic invertebrate, rather than recalcitrant organic carbon, mineralogical features such as iron oxides, or phytoplankton.

MICROPHOTOMETRIC ASSESSMENT OF SPECTRAL ABSORPTION AND ITS POTENTIAL APPLICATION FOR CHARACTERIZATION OF HARMFUL ALGAL SPECIES

A comparison was made of microphotometric measurements and spectrophotometric measurements of particulate spectral absorption of four algal species, including the chlorophyte Dunaliella tertiolecta Butcher; a nontoxic dinoflagellate, Amphidinium carterae Hulburt; a diatom, Chaetoceros gracilis Schütt; and a toxic dinoflagellate, Gymnodinium breve Steidinger. Particulate spectral absorption of monospecies cultures was estimated as the product of the average absorption efficiency factor, Qa, determined by microphotometry, the cellular cross‐sectional area, and the cell number density. Estimates of particulate spectral absorption from microphotometric measurements were, in most cases, within one standard deviation of values determined from spectrophotometric measurements of algal suspensions. Estimates of Qa(675) were shown to be consistent with values reported in previous studies for cells of similar size and pigmentation and were consistent with theoretical predictions. Absorption spectra of mixtures of C. gracilis and G. breve were numerically decomposed into contributions by absorption signatures of monospecies cultures using either spectrophotometric or microphotometric measurements as the basis for end members. Modeled contributions assigned to either species displayed trends consistent with the actual proportions contributed to the spectrum by each algal culture. However, the technique was sensitive to measurement variability, which reduced the level of agreement between modeled and actual contributions. The utility of this approach for identification of algal taxa will depend on the degree to which algal spectral absorption signatures differ and the capabilities for acquiring high‐resolution data with low signal‐to‐noise ratios.

A PSTTLRE-form of cdc2-like gene in the marine microalga Dunaliella tertiolecta.

To understand the genetic control of algal cell division cycle that pertains to phytoplankton bloom dynamics in the sea, we cloned and analyzed a gene coding for a cyclin-dependent kinase (CDK) for the chlorophyte Dunaliella tertiolecta. The cDNA cloned, 1061 bp long, contained an open reading frame of 314 amino acids. FASTA and GAP analyses showed that this sequence was most homologous to cdc2 out of all known cdks, with an identity of 54-68% and a similarity of 65-76% to cdc2 in higher plants, animals, and yeast. Several signature domains of cdc2 were identified from this sequence, although the PSTAIRE and GDSEID motifs were replaced with PSTTLRE and GDCELQ, respectively. Southern blot hybridization demonstrated that this gene occurred as a single copy in this species, and quantitative RT-PCR showed that the transcription of this gene was constitutive. The present results suggest that the universal cdc2 is conserved in the lower eukaryote with unique structural characteristics.

The significance of feeding history on the value of heterotrophic microzooplankton as prey for larval crabs

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 186:219-225 (1999) - doi:10.3354/meps186219 The significance of feeding history on the value of heterotrophic microzooplankton as prey for larval crabs Stephen D. Sulkin*, Gene L. McKeen Shannon Point Marine Center, 1900 Shannon Point Road, Anacortes, Washington 98221, USA *E-mail: sulkin@cc.wwu.edu ABSTRACT: Larvae of 4 species of crabs of the genus Cancer (C. gracilis, C. magister, C. oregonensis and C. productus) were raised in the laboratory from hatching to the post-larval megalopa stage on diets of nauplii of the brine shrimp Artemia sp. and on 2 diets of the rotifer Brachionus plicatilis. One of the rotifer diets had been cultured on the chlorophyte Dunaliella tertiolecta, the other on the chrysophyte Isochrisis galbana. In 3 of the 4 crab species, Dunaliella-fed rotifers did not support development to the megalopa, whereas Isochrisis-fed rotifers did. In the fourth species, C. productus, survival to the megalopa was only 1% on Dunaliella-fed rotifers, whereas Isochrisis-fed rotifers supported development equal to that of the Artemia-fed control. Differences between the 2 rotifer diets were evident in all species by Stage 3, although the first significant occurrence of increased mortality on the Dunaliella-fed rotifer diet varied among species. In all 4 species, development rate was faster as early as the first molt on the Isochrisis-fed rotifer diet as compared to the Dunaliella-fed rotifer diet, a difference maintained throughout development. Only in C. gracilis did survival to the megalopa on the Isochrisis-fed rotifer fall substantially below that seen for Artemia-fed larvae. Stage mortality on the Isochrisis-fed rotifer diet typically equaled that of the Artemia sp. control. Differences in development rates between the Isochrisis-fed rotifer diet and the Artemia sp. diet varied among species, as did megalopa weight. We attribute differences between the 2 rotifer diets to the high content of long-chain ω-3 polyunsaturated fatty acids in I. galbana and their absence in D. tertiolecta. The value of heterotrophic microzooplankton as prey for crab larvae, either in culture or in nature, will depend upon the prey's own feeding history. KEY WORDS: Crab larvae · Diet · Heterotrophs · Microzooplankton Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 186. Publication date: September 17, 1999 Print ISSN:0171-8630; Online ISSN:1616-1599 Copyright © 1999 Inter-Research.

Electron microscopy of the marine microalga Dunaliella tertiolecta exposed to triphenyltin

Chemostat-grown cells of the chlorophyte Dunaliella tertiolecta (Butcher) exposed to triphenyltin were examined using transmission electron microscopy. Following a 1-h exposure to 21 and 84μM triphenyltin, mitochondria underwent structural damage and the thylakoid membranes of a small proportion of cells spread from the usual compact arrangement. Prolonging the exposure time resulted in significant cell lysis in cultures exposed to 84μM triphenyltin.

Physiological stress and cell death in marine phytoplankton: Induction of proteases in response to nitrogen or light limitation

The physiological processes of natural phytoplankton mortality due to environmental stress (vs. that caused by sedimentation and predation) are poorly understood. Cell survival was examined in batch cultures of the diatom Thalassiosira weissflogii and the chlorophyte Dunaliella tertiolecta during deprivation of fixed nitrogen or light. Despite severe impairment of photosynthetic efficiencies, both species remained viable during 2 weeks of N starvation. Under N stress, a specific protease was induced in the diatom, overall activity of proteases doubled, and there was gradual, selective loss of certain proteins, especially ribulose 1,5‐bisphosphate carboxylase/oxygenase. Light‐deprived diatoms were virtually unaffected, but the chlorophyte underwent catastrophic cell death after about 6 d of darkness. Cell death coincided with a large increase in protease activity and the induction of a specific protease. Although we cannot completely rule out roles for viruses or bacteria in the losses of cells, the consistent timing, the unique response to stress, and the coincident expression of a specific protease strongly suggest that the process is a form of autocatalyzed cell death, such as apoptosis. While of uncertain adaptive significance, phytoplankton cell death may have implications for species succession and cycling of organic matter in aquatic ecosysterns.

Rubisco of Dunaliella tertiolecta is redistributed between the pyrenoid and the stroma as a light?/shade response

We used an immunofluorescence technique to investigate the effects of varying light regimes on ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) in individual cells of the chlorophyte Dunaliella tertiolecta Butcher. These studies were carried out between September 1995 and February 1996. The population was heterogeneous with respect to Rubisco localization in the pyrenoid, i.e. in some cells Rubisco staining was highly concentrated in the pyrenoid while in others it was evenly distributed throughout the chloroplast stroma. When light intensity was varied sevenfold, the fraction of the cell population that displayed distinct Rubisco staining in the pyrenoid (PR-index) was correlated with light intensity, although the average Rubisco abundance per cell or per total cellular protein appeared fairly constant. In darkness, or when treated with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), the PR-index decreased markedly during the first 4 and 3 h, respectively, and then remained at low levels, while the cell division cycle progression remained unaffected. We conclude that D. tertiolecta probably possesses an adaptive mechanism, i.e. the redistribution of Rubisco between the pyrenoid (probably the active site of Rubisco activation and CO2 fixation) and the stroma (probably a reservoir of deactivated Rubisco that is readily available for transport to the pyrenoid and activation), to respond to variations in irradiance or photosynthetic inhibition. Our results also suggest that this mechanism is insensitive to slight variations in growth irradiance and to seasonal changes in photoperiod and temperature.

Pyrenoid localization of Rubisco in relation to the cell cycle and growth phase of Dunaliella tertiolecta (Chlorophyceae)

Localization of ribulose 1,5-bisphosphate carboxylase/oxygenase in pyrenoids of the chlorophyte Dunaliella tertiolecta Butcher was analyzed using immunofluorescence. Some cells displayed distinct pyrenoid-Rubisco staining (i.e. Rubisco was highly concentrated in the pyrenoid), whereas others displayed weak staining throughout the chloroplast stroma. Paired pyrenoid-Rubisco units were observed, suggesting that pyrenoid division had occurred. The percentage of cells having distinct pyrenoid-Rubisco staining (PR-index) was depressed drastically by darkness, but the cell cycle, which was monitored by immunofluorescence of the proliferating cell nuclear antigen (PCNA), was not affected. Inversely, neither the cell cycle inhibitors hydroxyurea and colchicine nor short-term nutrient depletion affected the PR-index. These results, along with the observation that cells with or without distinct pyrenoid-Rubisco staining had similar DNA contents, suggest that variation in pyrenoid localization of Rubisco was not cell cycle dependent. In addition, the PR-index increased along with growth rates in the early exponential growth phase and decreased to zero in the late stationary phase. After a stationary culture was diluted by fresh medium, the PR-index increased from 0 to high levels within 24 hours. These results suggest that pyrenoid-localization of Rubisco is associated with the active growth phase.