John A. Berges

The Phaeodactylum genome reveals the evolutionary history of diatom genomes.

Diatoms are photosynthetic secondary endosymbionts found throughout marine and freshwater environments, and are believed to be responsible for around one-fifth of the primary productivity on Earth. The genome sequence of the marine centric diatom Thalassiosira pseudonana was recently reported, revealing a wealth of information about diatom biology. Here we report the complete genome sequence of the pennate diatom Phaeodactylum tricornutum and compare it with that of T. pseudonana to clarify evolutionary origins, functional significance and ubiquity of these features throughout diatoms. In spite of the fact that the pennate and centric lineages have only been diverging for 90 million years, their genome structures are dramatically different and a substantial fraction of genes (∼40%) are not shared by these representatives of the two lineages. Analysis of molecular divergence compared with yeasts and metazoans reveals rapid rates of gene diversification in diatoms. Contributing factors include selective gene family expansions, differential losses and gains of genes and introns, and differential mobilization of transposable elements. Most significantly, we document the presence of hundreds of genes from bacteria. More than 300 of these gene transfers are found in both diatoms, attesting to their ancient origins, and many are likely to provide novel possibilities for metabolite management and for perception of environmental signals. These findings go a long way towards explaining the incredible diversity and success of the diatoms in contemporary oceans.

EVOLUTION OF AN ARTIFICIAL SEAWATER MEDIUM: IMPROVEMENTS IN ENRICHED SEAWATER, ARTIFICIAL WATER OVER THE LAST TWO DECADES

Although most phycologists use natural seawater for culturing marine species, artificial media continue to play important roles in overcoming problems of supply and seasonal variability in the quality of natural seawater and also for experiments involving manipulation of micro‐ and macronutrients. Several artificial media have been developed over the last 90 years; enriched seawater, artificial water (ESAW) is among the more popular recipes. ESAW has the advantage of an ionic balance that is somewhat closer to that of normal seawater. The original paper compared the growth of 83 strains of microalgae in natural seawater (ESNW) versus ESAW and determined that 23% grew more poorly in the artificial water. Since 1980, however, the composition of ESAW, as used by the original authors, has changed considerably. In particular, the added forms of phosphate, iron, and silicate have been changed and the trace metal mixture has been altered to include nickel, molybdenum, and selenium. We tested whether these changes improved the ability of the artificial medium to grow previously difficult to grow phytoplankton species. To test this, we selected eight species that had been shown to grow better in ESNW than in ESAW and compared their growth again, using the currently used recipe with all the above modifications. For all but one species (Apedinella spinifera), growth rate and final yield was no different between the media but in one case (Emiliania huxleyi) was slightly higher in ESAW. No differences in cell morphology or volume were found in any case. We conclude that changes to the enrichment portion of the recipe have significantly improved this artificial seawater medium and that it can be used to grow an even wider range of coastal and open ocean species.

Differential Effects of Nitrogen Limitation on Photosynthetic Efficiency of Photosystems I and II in Microalgae.

The effects of nitrogen starvation on photosynthetic efficiency were examined in three unicellular algae by measuring changes in the quantum yield of fluorescence with a pump-and-probe method and thermal efficiency (i.e. the percentage of trapped energy stored photochemically) with a pulsed photoacoustic method together with the inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea to distinguish photosystems I and II (PSI and PSII). Measured at 620 nm, maximum thermal efficiency for both photosystems was 32% for the diatom Thalassiosira weissflogii (PSII:PSI ratio of 2:1), 39% for the green alga Dunaliella tertiolecta (PSII:PSI ratio of 1:1), and 29% for the cyanobacterium Synechococcus sp. PCC 7002 (PSII:PSI ratio of 1:2). Nitrogen starvation decreased total thermal efficiency by 56% for T. weissflogii and by 26% for D. tertiolecta but caused no change in Synechococcus. Decreases in the number of active PSII reaction centers (inferred from changes in variable fluorescence) were larger: 86% (T. weissflogii), 65% (D. tertiolecta), and 65% (Synechococcus). The selective inactivation of PSII under nitrogen starvation was confirmed by independent measurements of active PSII using oxygen flash yields and active PSI using P700 reduction. Relatively high thermal efficiencies were measured in all three species in the presence of the PSII inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, suggesting the potential for significant cyclic electron flow around PSI. Fluorescence or photoacoustic data agreed well; in T. weissflogii, the functional cross-sectional area of PSII at 620 nm was estimated to be the same using both methods (approximately 1.8 x 102 A2). The effects of nitrogen starvation occur mainly in PSII and are well represented by variable fluorescence measurements.

Cell Death in the Unicellular Chlorophyte Dunaliella tertiolecta. A Hypothesis on the Evolution of Apoptosis in Higher Plants and Metazoans

Apoptosis is essential for normal growth and development of multicellular organisms, including metazoans and higher plants. Although cell death processes have been reported in unicellular organisms, key elements of apoptotic pathways have not been identified. Here, we show that when placed in darkness, the unicellular chlorophyte alga Dunaliella tertiolecta undergoes a form of cell death reminiscent of apoptosis in metazoans. Many morphological criteria of apoptotic cell death were met, including an increase in chromatin margination, degradation of the nucleus, and DNA fragmentation. Biochemical assays of the activities of cell death-associated proteases, caspases, measured using highly specific fluorogenic substrates, increased with time in darkness and paralleled the morphological changes. The caspase-like activities were inhibited by caspase-specific inhibitors. Antibodies raised against mammalian caspases cross-reacted with specific proteins in the alga. The pattern of expression of these immunologically reactive proteins was correlated with the onset of cell death. The occurrence of key components of apoptosis, and particularly a caspase-mediated cell death cascade in a relatively ancient linage of eukaryotic photoautotrophs, argues against current theories that cell death evolved in multicellular organisms. We hypothesize that key elements of cell death pathways were transferred to the nuclear genome of early eukaryotes through ancient viral infections in the Precambrian Ocean before the evolution of multicellular organisms and were subsequently appropriated in both metazoan and higher plant lineages.

What is the role and nature of programmed cell death in phytoplankton ecology

Cell death is a fundamental process in all metazoan organisms. In contrast, the ecological role of cell death in phytoplankton has been sorely neglected: the causes and biochemistry of cell death, and the quantitative significance of cell death in the ecology of phytoplankton populations and in broader biogeochemical cycles, are not well understood. Metazoan cell death is much better described, due to its accepted roles in the regulation of multicellular life. In metazoan cells, an influential paradigm suggests that there are two morphological outcomes of cell death, one caused by an ‘active’ pathway within the cell (so-called ‘programmed cell death’; PCD), and the other from a ‘passive’ externally-driven process (necrosis). Here, we examine the development of this paradigm, and associated concepts, in plant, animal, and microbial life, and discuss the role of cell death amongst the diverse taxa of the phytoplankton. Several recent studies suggest PCD operates in cyanobacteria, chlorophytes, and dinoflagellates. A better understanding of phytoplankton cell death will potentially provide insight into bloom development, intercellular signalling and population regulation. Understanding the role of PCD in phytoplankton life-history will likely come through examination of metabolic differentiation within phytoplankton populations, of which at present there are only isolated reports. Although bacterial metabolic differentiation (e.g. in the formation of biofilms) is well accepted, metabolic differentiation and group selection amongst microalgae are poorly understood, and are ideas which merit greater research effort. If a process similar to metazoan PCD is widespread amongst unicellular algae, then a rethinking of the ecological relationships between and within phytoplankton populations will be necessary. We highlight the semantic difficulties present in this relatively new field of study and make recommendations for future study.

Integrated Photo-Bioelectrochemical System for Contaminants Removal and Bioenergy Production

An integrated photobioelectrochemical (IPB) system was developed by installing a microbial fuel cell (MFC) inside an algal bioreactor. This system achieves the simultaneous removal from a synthetic solution of organics (in the MFC) and nutrients (in the algal bioreactor), and the production of bioenergy in electricity and algal biomass through bioelectrochemical and microbiological processes. During the one-year operation, the IPB system removed more than 92% of chemical oxygen demand, 98% of ammonium nitrogen, and 82% of phosphate and produced a maximum power density of 2.2 W/m(3) and 128 mg/L of algal biomass. The algal growth provided dissolved oxygen to the cathode reaction of the MFC, whereas electrochemical oxygen reduction on the MFC cathode buffered the pH of the algal growth medium (which was also the catholyte). The system performance was affected by illumination and dissolved oxygen. Initial energy analysis showed that the IPB system could theoretically produce enough energy to cover its consumption; however, further improvement of electricity production is desired. An analysis of the attached and suspended microbes in the cathode revealed diverse bacterial taxa typical of aquatic and soil bacterial communities with functional roles in contaminant degradation and nutrient cycling.

Mortality in cultures of the dinoflagellate Amphidinium carterae during culture senescence and darkness

The study of cell death in higher plants and animals has revealed the existence of an active (‘programmed’) process in most types of cell, and similarities in cell death between plants, animals, yeast and bacteria suggest an evolutionarily ancient origin of programmed cell death (PCD). Despite their global importance in primary production, information on algal cell death is limited. Algal cell death could have similarities with metazoan cell death. One morphotype of metazoan PCD, apoptosis, can be induced by light deprivation in the unicellular chlorophyte Dunaliella tertiolecta. The situation in other algal taxa is less clear. We used a model dinoflagellate (Amphidinium carterae) to test whether mortality during darkness and culture senescence showed apoptotic characteristics. Using transmission electron microscopy, fluorescent biomarkers, chlorophyll fluorescence and particulate carbon analysis we analysed the process of cell mortality and found that light deprivation caused mass mortality. By contrast, fewer dead cells (5–20% of the population) were found in late–phase cultures, while a similar degenerate cell morphology (shrunken, chlorotic) was observed. On morphological grounds, our observations suggest that the apoptotic cell death described in D. tertiolecta does not occur in A. carterae. Greater similarity was found with paraptosis, a recently proposed alternative morphotype of PCD. A paraptotic conclusion is supported by inconclusive DNA fragmentation results. We emphasize the care that must be taken in transferring fundamental paradigms between phylogenetically diverse cell types and we argue for a greater consistency in the burden of proof needed to assign causality to cell death processes.

A comparison of Lowry, Bradford and Smith protein assays using different protein standards and protein isolated from the marine diatom Thalassiosira pseudonana

Two spectrophotometric assays for protein commonly used in marine research (Coomassie stain, “Bradford”; alkaline copper, “Lowry”) and a more recent assay which has not been applied in this field (bicinchoninic acid, “Smith”) were compared for homogenates of the marine diatom Thalassiosira pseudonona using bovine serum albumin (BSA) as a standard. When homogenates were prepared by precipitating protein with trichloroacetic acid (TCA) and redissolving in 1 N NaOH, the protein content estimated by the Lowry and Smith assays agreed closely, but was consistently 20% higher than that indicated by the Bradford assay. To determine if this difference was due to the choice of a protein standard, protein from T. pseudonana was purified and compared to BSA, bovine gamma-globulin (BGG), and casein. The reactivity of the purified protein (expressed as the slope of the absorbance vs protein concentration curve) did not differ between cultures grown at high or low irradiance. For the Smith and Bradford assays the reactivity of BSA was not significantly different from algal protein, but for the Lowry assay, algal protein was significantly higher in reactivity than BSA. BGG was not significantly different in reactivity from algal protein for the Lowry and Smith assays, but BGG gave significantly lower absorbances than algal protein in the Bradford assay. These results suggest that BSA is a suitable standard for algal protein in the Bradford assays, while BGG is preferable for the Lowry assay. Either protein standard could be used for the Smith assay. Differences in purified algal protein reactivity compared to BSA could not account for the differences among the assays, nor could interference by chlorophyll a. Precipitating protein with TCA prior to analyses gave lower protein than direct analyses of homogenates for the Lowry and Smith assays, but no differences were found for the Bradford assay. As a result, the Lowry and Smith assays indicated up to 60% greater protein than the Bradford if TCA precipitation was not performed. This may be due to removal of free amino acids and small peptides which are less reactive in the Bradford assay. The 20% higher protein found in the Lowry or Smith vs Bradford assays may be due to different assay sensitivity to small peptides or other compounds which are precipitated along with proteins by TCA. Although the Smith assay is substantially simpler to perform than the Lowry, there appear to be no quantitative differences in the results. It remains unclear which spectrophotometric assay is most accurate, but the Bradford assay is faster and simpler, and is less likely to be affected by non-protein compounds found in marine phytoplankton.

Miniview: Algal nitrate reductases

The past decade has seen substantial breakthroughs in understanding the biochemistry, molecular biology and regulation of nitrate reductases (NR) in higher plants and green algae. In contrast, although there has been considerable interest in using various measurements of NR to provide ecophysiological information, comparable knowledge of NR is largely lacking in algal groups other than chlorophytes. Applying information about NR from chlorophytes and higher plants to other algae may be difficult. There is evidence that nonchlorophyte forms of NR are diverse and distinct in terms of biochemical characteristics and regulatory features. Key areas to be pursued in non-chlorophyte algae include the identification and adoption of model organisms for NR research in different algal groups; the creation of selected and engineered mutants; the purification, biochemical characterization and production of antibodies to different algal forms of NR; the identification of NR genes; and the undertaking of coordinated res...

DIEL PERIODICITY OF NITRATE REDUCTASE ACTIVITY AND PROTEIN LEVELS IN THE MARINE DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)

The diel variation and regulation of the enzyme nitrate reductase (NR) were examined in the diatom Thalassiosira weissflogii (Gru.) Fryxell et Hasle. NR was purified, and polyclonal antibodies were raised to a 98‐kD polypeptide. The antibodies cross‐reacted only with proteins from closely related diatom species, suggesting significant epitopic variation of this enzyme within algal divisions. Neither NR enzymatic activity nor protein was detected in cells grown with ammonium as the sole nitrogen source; the addition of ammonium to cells growing on nitrate decreased both protein levels and enzyme activity by 40% within 2 h. In cells grown on a 12:12 h LD cycle, NR activity and NR protein levels were highly correlated, with a peak at midday, a decrease toward the end of the photoperiod, and an increase in activity beginning near the end of the dark period. The addition of actinomycin D (an inhibitor of RNA synthesis) and cycloheximide (an inhibitor of protein synthesis) affected NR activity and NR protein levels identically, strongly suggesting that this nuclear‐encoded protein is regulated primarily at a transcriptional level. The diel pattern of NR protein and activity ceased immediately following transfer to continuous light, indicating that the periodicity is not directly controlled by a circadian rhythm. Time‐lagged cross‐correlation analysis revealed a 6‐h phased difference between the minimum enzyme activity or protein levels and the maximum cellular carbon pool. On the basis of the experimental results, we develop a model proposing that (1) NR activity is regulated primarily by transcriptional regulation of NR synthesis and that (2) the level of expression of the enzyme during a given day is correlated with the integrated pool of organic carbon accumulated during the preceding photoperiod.