Genome-wide analysis of the diatom cell cycle unveils a novel type of cyclins involved in environmental signaling

Background

Despite the enormous importance of diatoms in aquatic ecosystems and their broad industrial potential, little is known about their life cycle control. Diatoms typically inhabit rapidly changing and unstable environments, suggesting that cell cycle regulation in diatoms must have evolved to adequately integrate various environmental signals. The recent genome sequencing of Thalassiosira pseudonana and Phaeodactylum tricornutum allows us to explore the molecular conservation of cell cycle regulation in diatoms.

Results

By profile-based annotation of cell cycle genes, counterparts of conserved as well as new regulators were identified in T. pseudonana and P. tricornutum. In particular, the cyclin gene family was found to be expanded extensively compared to that of other eukaryotes and a novel type of cyclins was discovered, the diatom-specific cyclins. We established a synchronization method for P. tricornutum that enabled assignment of the different annotated genes to specific cell cycle phase transitions. The diatom-specific cyclins are predominantly expressed at the G1-to-S transition and some respond to phosphate availability, hinting at a role in connecting cell division to environmental stimuli.

Conclusion

The discovery of highly conserved and new cell cycle regulators suggests the evolution of unique control mechanisms for diatom cell division, probably contributing to their ability to adapt and survive under highly fluctuating environmental conditions.     

Bacterial diketopiperazines stimulate diatom growth and lipid accumulation

Diatoms are photosynthetic microalgae that fix a significant fraction of the world’s carbon. Because of their photosynthetic efficiency and high-lipid content, diatoms are priority candidates for biofuel production. Here, we report that sporulating Bacillus thuringiensis and other members of the Bacillus cereus group, when in co-culture with the marine diatom Phaeodactylum tricornutum, significantly increase diatom cell count. Bioassay-guided purification of the mother cell lysate of B. thuringiensis led to the identification of two diketopiperazines (DKPs) that stimulate both P. tricornutum growth and increase its lipid content. These findings may be exploited to enhance P. tricornutum growth and microalgae-based biofuel production. As increasing numbers of DKPs are isolated from marine microbes, the work gives potential clues to bacterial-produced growth factors for marine microalgae.     

The ancestors of diatoms evolved a unique mitochondrial dehydrogenase to oxidize photorespiratory glycolate

Like other oxygenic photosynthetic organisms, diatoms produce glycolate, a toxic intermediate, as a consequence of the oxygenase activity of Rubisco. Diatoms can remove glycolate through excretion and through oxidation as part of the photorespiratory pathway. The diatom Phaeodactylum tricornutum encodes two proteins suggested to be involved in glycolate metabolism: PtGO1 and PtGO2. We found that these proteins differ substantially from the sequences of experimentally characterized proteins responsible for glycolate oxidation in other species, glycolate oxidase (GOX) and glycolate dehydrogenase. We show that PtGO1 and PtGO2 are the only sequences of P. tricornutum homologous to GOX. Our phylogenetic analyses indicate that the ancestors of diatoms acquired PtGO1 during the proposed first secondary endosymbiosis with a chlorophyte alga, which may have previously obtained this gene from proteobacteria. In contrast, PtGO2 is orthologous to an uncharacterized protein in Galdieria sulphuraria, consistent with its acquisition during the secondary endosymbiosis with a red alga that gave rise to the current plastid. The analysis of amino acid residues at conserved positions suggests that PtGO2, which localizes to peroxisomes, may use substrates other than glycolate, explaining the lack of GOX activity we observe in vitro. Instead, PtGO1, while only very distantly related to previously characterized GOX proteins, evolved glycolate-oxidizing activity, as demonstrated by in gel activity assays and mass spectrometry analysis. PtGO1 localizes to mitochondria, consistent with previous suggestions that photorespiration in diatoms proceeds in these organelles. We conclude that the ancestors of diatoms evolved a unique alternative to oxidize photorespiratory glycolate: a mitochondrial dehydrogenase homologous to GOX able to use electron acceptors other than O₂.     

Comparison of oligomeric states and polypeptide compositions of fucoxanthin chlorophyll a/c-binding protein complexes among various diatom species

Fucoxanthin chlorophyll a/c-binding protein (FCP) is a unique light-harvesting apparatus in diatoms. Several biochemical characteristics of FCP oligomer and trimer from different diatom species have been reported previously. However, the integration of information about molecular organizations and polypeptides of FCP through a comparison among diatoms has not been published. In this study, we used two-dimensional clear-native/SDS-PAGE to compare the oligomeric states and polypeptide compositions of FCP complexes from four diatoms: Chaetoceros gracilis, Thalassiosira pseudonana, Cyclotella meneghiniana, and Phaeodactylum tricornutum. FCP oligomer was found in C. gracilis, T. pseudonana, and C. meneghiniana, but not in P. tricornutum. The oligomerization varied among the three diatoms, although a predominant subunit having similar molecular weight was recovered in each FCP oligomer. These results suggest that the predominant subunit is involved in the formation of high FCP oligomerization in each diatom. In contrast, FCP trimer was found in all the diatoms. The trimerizations were quite similar, whereas the polypeptide compositions were markedly different. On the basis of this information and that from mass spectrometric analyses, the gene products in each FCP complex were identified in T. pseudonana and P. tricornutum. Based on these results, we discuss the role of FCP oligomer and trimer from the four diatoms.     

Ovothiols as free-radical scavengers and the mechanism of ovothiol-promoted NAD(P)H-O2 oxidoreductase activity

Racemic ovothiol A [(+/-)-1a] and the ovothiol model compound 1,5-dimethyl-4-mercaptoimidazole (DMI, 2) were found to scavange the free radicals Fremy's salt (4) and Banfield' radical (5) much more rapidly than did the thiol antioxidant glutathione. Ovothiol A also scavenges the tyrosyl radical, with efficiency comparable to that of ascorbic acid and the tocopherol analogue trolox (3). The ovothiol model compound DMI was found to scavenge superoxide with a rate constant comparable to that of the reaction between superoxide and glutathione. These results suggest both a free-radical scavenging role for the ovothiols and a mechanism by which the ovothiols confer NAD(P)H-O2 oxidoreductase activity upon the enzyme ovoperoxidase. Investigation of this mechanism implicates the ovothiol thiyl radical and the NAD radical as key intermediates. The ovothiyl radical appears to be unreactive toward oxygen but highly reactive toward NADH. An estimate of the one-electron oxidation potential of the ovothiol anion is presented. The physical basis for the stability of the ovothiol free radical is discussed.     

Ovothiols, a family of redox-active mercaptohistidine compounds from marine invertebrate eggs

We have previously reported a novel thiol compound, 1-methyl-N alpha,N alpha-dimethyl-4-mercaptohistidine, or ovothiol, present at high concentration in the eggs of the sea urchin Strongylocentrotus purpuratus [Turner, E., Klevit, R., Hopkins, P. B., & Shapiro, B. M. (1986) J. Biol. Chem. 261, 13056-13063]. Here we report two related compounds, 1-methyl-N alpha-methyl-4-mercaptohistidine, or ovothiol B, from the scallop Chlamys hastata, and 1-methyl-4-mercaptohistidine, or ovothiol A, from the starfish Evasterias troschelii. These two compounds, as well as the S. purpuratus compound now designated ovothiol C, were isolated from eggs or ovarian tissue by S-carboxymethylation with [3H]iodoacetic acid, ion-exchange chromatography and ion-pairing high-pressure liquid chromatography. The structures of S-(carboxymethyl)ovothiols A and B were determined by 1H NMR, and that of ovothiol A was confirmed by comparison with authentic methylhistidine samples after desulfuration with Raney nickel. In the ovary of each species, the predominant methylation form of ovothiol accounts for at least 80% of the total 4-mercaptohistidine. The ovothiol concentration of the ovary far exceeds that of the testis or somatic tissues. The ovothiol C content of unfertilized S. purpuratus eggs is 1.14 mumol/10(6) eggs, equivalent to approximately 4.3 mM average concentration; the glutathione (GSH + GSSG) content is 0.9 mumol/10(6) eggs. In this species, high ovothiol levels persisted for the first 2 weeks of embryonic development. Ovothiol and glutathione account for virtually all of the trichloroacetic acid soluble-SH groups in the egg; these results are compared to several previous studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of iron on the growth of Phaeodactylum tricornutum via photosynthesis

Iron is a limiting factor that controls the phytoplankton biomass in the modern ocean, and iron fertilization of the ocean could lead to blooms dominated by diatoms. Thus, iron plays an important role in controlling the distribution of diatoms. In this study, we measured the growth rate and photosynthetic activity of the model diatom Phaeodactylum tricornutum cultured under different iron concentrations and found that it grew more rapidly and had a much higher photosynthetic efficiency under higher iron concentrations. In order to explore the unique mechanism of the response of diatoms to iron, a proteomic analysis was carried out, and the results indicated that iron promotes the Calvin cycle of P. tricornutum. Diatoms can tolerate the pressure of iron limitation by replacing iron‐rich proteins with flavodoxin, and so on. Moreover, we found that the photosystem I (PSI) activity of iron‐limited algae that were treated by N’,N’,N’,N’‐tetramethyl‐p‐phenylenediamine (TMPD) was increased significantly. As TMPD plays the role of a cytochrome b₆/f complex that transfers electrons from photosystem II to PSI, the cytochrome b₆/f complex is the key to photosynthesis regulation. Iron could influence the growth of P. tricornutum by regulating its biosynthesis. All of the results suggest that iron might affect the growth of diatoms through the Calvin cycle and the cytochrome b₆/f complex.     

The harpacticoid copepod Tisbe holothuriae is resistant to the insidious effects of polyunsaturated aldehyde-producing diatoms

Numerous species of diatoms liberate oxylipins including polyunsaturated aldehydes (PUAs) in response to cellular damage such as may occur during grazing. PUAs are cyto- and genotoxic and negatively disrupt reproductive processes in copepods, their principal grazers, although experimental evidence would suggest that the grazer response may be species specific. The reproduction of the benthic harpacticoid copepod Tisbe holothuriae was compared over two generations. Copepods were reared using four diet treatments: PUA-producing diatom strains Skeletonema marinoi (Adriatic Sea Isolate FE6) and Melosira nummuloides (CCAP 1048/6); and non-PUA-producing diatom strains Phaeodactylum tricornutum (CCAP 1052/A) and S. marinoi (Seasalter (Walney) Ltd). Life tables were generated for each treatment using measured reproductive parameters and the net reproductive rate (R₀) calculated. No significant differences were observed between the individual reproductive parameters of T. holothuriae fed PUA-producing diatoms compared to those fed non-PUA-producing diatoms although diets of P. tricornutum resulted in some decreases in individual reproductive parameters in the second generation. There were no significant differences in the R₀ values between the four tested diets. These observations indicate that T. holothuriae exhibits a tolerance of known PUA-producing diatom diets that has not been similarly demonstrated in pelagic calanoid copepods. Harpacticoid copepods may have a greater capacity to detoxify diatom oxylipins than their planktonic calanoid counterparts.     

Cold-induced [Ca2+]cyt elevations function to support osmoregulation in marine diatoms

Diatoms are a group of microalgae that are important primary producers in a range of open ocean, freshwater, and intertidal environments. The latter can experience substantial long- and short-term variability in temperature, from seasonal variations to rapid temperature shifts caused by tidal immersion and emersion. As temperature is a major determinant in the distribution of diatom species, their temperature sensory and response mechanisms likely have important roles in their ecological success. We examined the mechanisms diatoms use to sense rapid changes in temperature, such as those experienced in the intertidal zone. We found that the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana exhibit a transient cytosolic Ca²⁺ ([Ca²⁺]_cyt) elevation in response to rapid cooling, similar to those observed in plant and animal cells. However, [Ca²⁺]_cyt elevations were not observed in response to rapid warming. The kinetics and magnitude of cold-induced [Ca²⁺]_cyt elevations corresponded with the rate of temperature decrease. We did not find a role for the [Ca²⁺]_cyt elevations in enhancing cold tolerance but showed that cold shock induces a Ca²⁺-dependent K⁺ efflux and reduces mortality of P. tricornutum during a simultaneous hypo-osmotic shock. As intertidal diatom species may routinely encounter simultaneous cold and hypo-osmotic shocks during tidal cycles, we propose that cold-induced Ca²⁺ signaling interacts with osmotic signaling pathways to aid in the regulation of cell volume. Our findings provide insight into the nature of temperature perception in diatoms and highlight that cross-talk between signaling pathways may play an important role in their cellular responses to multiple simultaneous stressors.

A calcium signaling pathway in marine diatoms is activated by cold temperature and enhances survival during simultaneous hypo-osmotic stress.     

Kinetic exploration of nitrate-accumulating microalgae for nutrient recovery

Within sustainable resource management, the recovery of nitrogen and phosphorus nutrients from waste streams is becoming increasingly important. Although the use of microalgae has been described extensively in environmental biotechnology, the potential of nitrate-accumulating microalgae for nutrient recovery has not been investigated yet. The ability of these marine microorganisms to concentrate environmental nitrate within their biomass is remarkable. The aim of this study was to investigate the application potential of nitrate-accumulating diatoms for nutrient recovery from marine wastewaters. The intracellular nitrate storage capacity was quantified for six marine diatom strains in synthetic wastewater. Amphora coffeaeformis and Phaeodactylum tricornutum stored the highest amount of nitrate with respectively 3.15 and 2.10 g N L^?1 of cell volume, which accounted for 17.3 and 4.6 %, respectively, of the total nitrogen content. The growth and nitrate and phosphate uptake of both diatoms were further analyzed and based on these features P. tricornutum showed the highest potential for nutrient recovery. A mathematical model was developed which included intracellular nitrate storage and the kinetic parameters were derived for P. tricornutum. Furthermore, a simulation study was performed to compare the performance of a proposed microalgal nutrient recovery unit with a conventional denitrification system for marine wastewater treatment. Overall, this study demonstrates the potential application of P. tricornutum for saline wastewater treatment with concurrent nitrogen and phosphorus recycling.     

Short-term impacts of polyunsaturated aldehyde-producing diatoms on the harpacticoid copepod, Tisbe holothuriae

Diatoms that produce toxic oxylipins can be detrimental to the reproductive success of aquatic invertebrates. Despite the potential importance of these toxins in shaping aquatic ecosystems, marine studies to date have focused almost exclusively on planktonic calanoid copepods. The current work examines the response of the benthic harpacticoid copepod, Tisbe holothuriae, to direct exposure to diatom-derived oxylipins and the short-term impact of oxylipin-producing diatom diets on reproductive success. The most toxic oxylipin was the polyunsaturated aldehyde (PUA) 2E,4E-decadienal with an LD₅₀ of 9.3 μM for T. holothuriae nauplii. The least tolerant life-stage was the nauplius followed by adult males then adult females. Short-term exposure to PUA-producing diatoms (Skeletonema marinoi and Melosira nummuloides) in maternal diets had no significant impact on reproductive success compared with non-PUA-producing diets (Skeletonema costatum, Navicula hanseni, Phaeodactylum tricornutum and Tetraselmis suecica). The PUA producers had no negative impact on the survival and development of naupliar stages to adulthood. T. holothuriae expresses a higher degree of tolerance to PUA-producing diatoms than many planktonic calanoids, possibly reflecting a degree of adaptation to higher stress levels associated with the benthos. This is the first study to investigate the reproductive responses of harpacticoid copepods feeding on known PUA-producing diatoms.     

A Rare Phaeodactylum tricornutum Cruciform Morphotype: Culture Conditions,Transformation and Unique Fatty Acid Characteristics

A rare Phaeodactylum tricornutum cruciform morphotype was obtained and stabilized with a proportion of more than 31.3% in L1 medium and is reported for the first time. Long-term culture and observation showed that the cruciform morphotype was capable of transforming to the oval form following the degeneration of arms by two processes. After three months of culture, four morphotypes existed in a relatively stable proportion in culture for six months (10.5% for oval, 11.3% for fusiform, 37.2% for triradiate and 41.0% for cruciform). Low temperature was particularly beneficial for cruciform cell formation. As the culture temperature decreased from 25°C to 10°C, the percentage of the cruciform morphotype increased from 39.1% to 55.3% approximately. The abundant cruciform cells endowed this strain with unique fatty acid characteristics. The strain cultured at 15°C showed both maximum content of neutral lipid in a single cell and total yield. The maximum content of fatty acid methyl esters was C16:1 for Phaeodactylum tricornutum cultured at four temperatures (43.82% to 50.82%), followed by C16:0 (20.47% to 22.65%). Unique fatty acid composition endowed this strain with excellent quality for biodiesel production.     

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.     

A model for carbohydrate metabolism in the diatom Phaeodactylum tricornutum deduced from comparative whole genome analysis

Background

Diatoms are unicellular algae responsible for approximately 20% of global carbon fixation. Their evolution by secondary endocytobiosis resulted in a complex cellular structure and metabolism compared to algae with primary plastids.

Methodology/Principal Findings

The whole genome sequence of the diatom Phaeodactylum tricornutum has recently been completed. We identified and annotated genes for enzymes involved in carbohydrate pathways based on extensive EST support and comparison to the whole genome sequence of a second diatom, Thalassiosira pseudonana. Protein localization to mitochondria was predicted based on identified similarities to mitochondrial localization motifs in other eukaryotes, whereas protein localization to plastids was based on the presence of signal peptide motifs in combination with plastid localization motifs previously shown to be required in diatoms. We identified genes potentially involved in a C4-like photosynthesis in P. tricornutum and, on the basis of sequence-based putative localization of relevant proteins, discuss possible differences in carbon concentrating mechanisms and CO₂ fixation between the two diatoms. We also identified genes encoding enzymes involved in photorespiration with one interesting exception: glycerate kinase was not found in either P. tricornutum or T. pseudonana. Various Calvin cycle enzymes were found in up to five different isoforms, distributed between plastids, mitochondria and the cytosol. Diatoms store energy either as lipids or as chrysolaminaran (a β-1,3-glucan) outside of the plastids. We identified various β-glucanases and large membrane-bound glucan synthases. Interestingly most of the glucanases appear to contain C-terminal anchor domains that may attach the enzymes to membranes.

Conclusions/Significance

Here we present a detailed synthesis of carbohydrate metabolism in diatoms based on the genome sequences of Thalassiosira pseudonana and Phaeodactylum tricornutum. This model provides novel insights into acquisition of dissolved inorganic carbon and primary metabolic pathways of carbon in two different diatoms, which is of significance for an improved understanding of global carbon cycles.     

Lysine biosynthesis and the evolution-of pennate marine diatoms

The classification of lysine biosynthetic pathways in various organisms have been used to investigate their descent in evolution. We have attempted these determinations in the diatoms Amphora coffeaeformis var:perpusilla (Grunow Cleve.) and Phaeodactylum tricornutum (Bohlin). Additionally, we have verified earlier results of Vogel in a green alga, Chlorella pyrenoidosa strain Tx 71105 (Texas Culture Collection). Our research indicates that the diaminopimelic acid route is involved in all three organisms. While these studies do not exclude the possible co-existence of the α-aminoadipic acid route, the results imply a closer evolutionary relationship of pennate diatoms to bacteria and “classical” photosynthetic plants rather than to heterotrophic or mixotrophic fungi and atypical algal strains such as the Euglenophyta.     

The intracellular distribution of inorganic carbon fixing enzymes does not support the presence of a C4 pathway in the diatom <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis>

Diatoms are unicellular algae and important primary producers. The process of carbon fixation in diatoms is very efficient even though the availability of dissolved CO₂ in sea water is very low. The operation of a carbon concentrating mechanism (CCM) also makes the more abundant bicarbonate accessible for photosynthetic carbon fixation. Diatoms possess carbonic anhydrases as well as metabolic enzymes potentially involved in C4 pathways; however, the question as to whether a C4 pathway plays a general role in diatoms is not yet solved. While genome analyses indicate that the diatom Phaeodactylum tricornutum possesses all the enzymes required to operate a C4 pathway, silencing of the pyruvate orthophosphate dikinase (PPDK) in a genetically transformed cell line does not lead to reduced photosynthetic carbon fixation. In this study, we have determined the intracellular location of all enzymes potentially involved in C4-like carbon fixing pathways in P. tricornutum by expression of the respective proteins fused to green fluorescent protein (GFP), followed by fluorescence microscopy. Furthermore, we compared the results to known pathways and locations of enzymes in higher plants performing C3 or C4 photosynthesis. This approach revealed that the intracellular distribution of the investigated enzymes is quite different from the one observed in higher plants. In particular, the apparent lack of a plastidic decarboxylase in P. tricornutum indicates that this diatom does not perform a C4-like CCM.     

The effect of high pH on structural lipids in diatoms

We tested the hypothesis that increased pH reduces the amount of structural lipids. To do this, we used three different diatoms (Phaeodactylum tricornutum CCAP strain, P. tricornutum TV strain and Amphiprora sp). We tested the effect of rapid increase from pH?7.5 to 10 by adding NaOH. The total lipid content was reduced by 13, 36 and 47 % in the P. tricornutum CCAP strain, TV strain and Amphiprora sp., respectively, 1 h after increasing the pH. The P. tricornutum CCAP strain was used for further testing the effect of pH on the lipid content during active growth. This strain was cultivated at pH?7.5 and 10, and the pH was regulated by the CO₂ inflow. The growth rate was similar (0.3 day^?1) in both pH treatments, but the lipid content in the pH?10 treatment was on average 28 % lower than in the pH?7.5 treatment. Our data support the hypothesis that structural lipids are reduced when pH increases to high levels. The results suggest that regulating the pH during algae cultivation could be used to refine the lipid composition in the harvested algal biomass.