Genome properties of the diatom Phaeodactylum tricornutum

Diatoms are a ubiquitous class of microalgae of extreme importance for global primary productivity and for the biogeochemical cycling of minerals such as silica. However, very little is known about diatom cell biology or about their genome structure. For diatom researchers to take advantage of genomics and post-genomics technologies, it is necessary to establish a model diatom species. Phaeodactylum tricornutum is an obvious candidate because of its ease of culture and because it can be genetically transformed. Therefore, we have examined its genome composition by the generation of approximately 1,000 expressed sequence tags. Although more than 60% of the sequences could not be unequivocally identified by similarity to sequences in the databases, approximately 20% had high similarity with a range of genes defined functionally at the protein level. It is interesting that many of these sequences are more similar to animal rather than plant counterparts. Base composition at each codon position and GC content of the genome were compared with Arabidopsis, maize (Zea mays), and Chlamydomonas reinhardtii. It was found that distribution of GC within the coding sequences is as homogeneous in P. tricornutum as in Arabidopsis, but with a slightly higher GC content. Furthermore, we present evidence that the P. tricornutum genome is likely to be small (less than 20 Mb). Therefore, this combined information supports the development of this species as a model system for molecular-based studies of diatom biology. The nucleotide sequence data reported has been deposited in GenBank Nucleotide Sequence Database (dbEST section) under accession nos. BI306757 through BI307753.     

Comparative genomics of the pennate diatom Phaeodactylum tricornutum

Diatoms are one of the most important constituents of phytoplankton communities in aquatic environments, but in spite of this, only recently have large-scale diatom-sequencing projects been undertaken. With the genome of the centric species Thalassiosira pseudonana available since mid-2004, accumulating sequence information for a pennate model species appears a natural subsequent aim. We have generated over 12,000 expressed sequence tags (ESTs) from the pennate diatom Phaeodactylum tricornutum, and upon assembly into a nonredundant set, 5,108 sequences were obtained. Significant similarity (E < 1E-04) to entries in the GenBank nonredundant protein database, the COG profile database, and the Pfam protein domains database were detected, respectively, in 45.0%, 21.5%, and 37.1% of the nonredundant collection of sequences. This information was employed to functionally annotate the P. tricornutum nonredundant set and to create an internet-accessible queryable diatom EST database. The nonredundant collection was then compared to the putative complete proteomes of the green alga Chlamydomonas reinhardtii, the red alga Cyanidioschyzon merolae, and the centric diatom T. pseudonana. A number of intriguing differences were identified between the pennate and the centric diatoms concerning activities of relevance for general cell metabolism, e.g. genes involved in carbon-concentrating mechanisms, cytosolic acetyl-Coenzyme A production, and fructose-1,6-bisphosphate metabolism. Finally, codon usage and utilization of C and G relative to gene expression (as measured by EST redundance) were studied, and preferences for utilization of C and CpG doublets were noted among the P. tricornutum EST coding sequences.     

三角褐指藻(Phaeodactylum tricornutum)通用转化载体的构建

三角褐指藻(Phaeodactylum tricornutum)是开展微藻生物柴油研究的理想材料。克隆了内源fcp基因簇的多个调控序列(启动子、终止子),构建了包括fcpB启动子-bar基因-fcpA终止子、以及fcpA启动子-多克隆位点(MCS)-fcpA终止子两个表达盒的通用转化载体pfcpA-MCS/fcpB-Bar,其特征是以bar基因作为选择标记,MCS区方便插入一至多个目的基因。新载体可用于三角褐指藻的重组蛋白表达、或油脂代谢相关基因的功能验证和代谢调控研究。     

Effect of starvation on the distribution of positional isomers and enantiomers of triacylglycerol in the diatom Phaeodactylum tricornutum

The diatom Phaeodactylum tricornutum was cultivated in a standard medium and under sulfur, silicon, nitrogen and phosphorus starvation and its triacylglycerols (TAGs) were analyzed by RP-HPLC/MS-APCI. Nearly 100 molecular species of polyunsaturated TAGs were identified. RP-HPLC was used to isolate positional isomers of TAGs, which were further separated by chiral HPLC. First eluted were those TAGs that have an eicosapentaenoic acid moiety in the sn-1 position. The ratios of symmetrical to asymmetrical TAGs in P. tricornutum were affected under sulfur-, nitrogen-, phosphorus- and silica-starvation, i.e. in cultivations involving cells in nutrient stress. The ratios of positional TAGs and also the proportions of enantiomers were changed. The ratios of symmetrical to asymmetrical TAGs in the control and under N- and P-starvation were very close. In the control, the ratio of 1,2-dipalmitoyl-3-eicosapentaenoyl-rac-glycerol to 1,3-dipalmitoyl-2-eicosapentaenoyl-rac-glycerol was 3:1 and the ratio of 1,2-dieicosapentaenoyl-3-palmitoyl-rac-glycerol to 1,3-dieicosapentaenoyl-2-palmitoyl-rac-glycerol was 9:1. Under N-starvation the ratios were reversed irrespective of the presence or absence of silicate in the medium. A similar pattern was found in P- and S-starvation.     

Inorganic-carbon uptake by the marine diatom Phaeodactylum tricornutum

Air-grown cells of the marine diatom Phaeodactylum tricornutum showed only 10% of the carbonic-anhydrase activity of air-grown Chlamydomonas reinhardtii. Measurement of carbonic-anhydrase activity using intact cells and cell extracts showed all activity was intracellular in Phaeodactylum. Photosynthetic oxygen evolution at constant inorganic-carbon concentration but varying pH showed that exogenous CO₂ was poorly utilized by the cells. Sodium ions increased the affinity of Phaeodactylum for HCO ₃ ^- and even at high HCO ₃ ^- concentrations sodium ions enhanced HCO ₃ ^- utilization. The internal inorganic-carbon pool (HCO ₃ ^- +CO₂] was measured using a silicone-oil-layer centrifugal filtering technique. The internal [HCO ₃ ^- +CO₂] concentration never exceeded 15% of the external [HCO ₃ ^- +CO₂] concentration even at the lowest external concentrations tested. It is concluded that an internal accumulation of inorganic carbon relative to the external medium does not occur in P. tricornutum.Abbreviation Hepes 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid     

Use of sodium bicarbonate to stimulate triacylglycerol accumulation in the chlorophyte Scenedesmus sp. and the diatom Phaeodactylum tricornutum

There is potential for algal-derived biofuel to help alleviate part of the world’s dependency on petroleum based fuels. However, research must still be done on strain selection, induction of triacylglycerol (TAG) accumulation, and fundamental algal metabolic studies, along with large-scale culturing techniques, harvesting, and biofuel/biomass processing. Here, we have advanced the knowledge on Scenedesmus sp. strain WC-1 by monitoring growth, pH, and TAG accumulation on a 14:10 light–dark cycle with atmospheric air or 5% CO₂ in air (v/v) aeration. Under ambient aeration, there was a loss of pH-induced TAG accumulation, presumably due to TAG consumption during the lower culture pH observed during dark hours (pH 9.4). Under 5% CO₂ aeration, the growth rate nearly doubled from 0.78 to 1.53 d^?1, but the pH was circumneutral (pH 6.9) and TAG accumulation was minimal. Experiments were also performed with 5% CO₂ during the exponential growth phase, which was then switched to aeration with atmospheric air when nitrate was close to depletion. These tests were run with and without the addition of 50 mM sodium bicarbonate. Cultures without added bicarbonate showed decreased growth rates with the aeration change, but there was no immediate TAG accumulation. The cultures with bicarbonate added immediately ceased cellular replication and rapid TAG accumulation was observed, as monitored by Nile Red fluorescence which has previously been correlated by gas chromatography to cellular TAG levels. Sodium bicarbonate addition (25 mM final concentration) was also tested with the marine diatom Phaeodactylum tricornutum strain Pt-1 and this organism also accumulated TAG.     

Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum

Research into diatom biology has now entered the post-genomics era, following the recent completion of the Thalassiosira pseudonana and Phaeodactylum tricornutum whole genome sequences and the establishment of Expressed Sequence Tag (EST) databases. The thorough exploitation of these resources will require the development of molecular tools to analyze and modulate the function of diatom genes in vivo. Towards this objective, we report here the identification of several reference genes that can be used as internal standards for gene expression studies by quantitative real-time PCR (qRT-PCR) in P. tricornutum cells grown over a diel cycle. In addition, we describe a series of diatom expression vectors based on Invitrogen Gateway technology for high-throughput protein tagging and overexpression studies in P. tricornutum. We demonstrate the utility of the diatom Destination vectors for determining the subcellular localization of a protein of interest and for immunodetection. The availability of these new resources significantly enriches the molecular toolbox for P. tricornutum and provides the diatom research community with well defined high-throughput methods for the analysis of diatom genes and proteins in vivo.     

Preparation of girdle lamella-containing chloroplasts from the diatom Phaeodactylum tricornutum

Chloroplasts were isolated from the diatom Phaeodactylum tricornutumby French press treatment and centrifugation. Electron micrographsof the isolated chloroplasts indicated that they lacked mostof the envelope membranes but retained the lamellar structurecharacteristic of the diatom chloroplast; three thylakoids weregrouped to form a band which transversed the chloroplast. Agirdle lamella also composed of three thylakoids surroundedthese transversal lamellae. The isolated chloroplasts were activein photosynthetic electron transport reactions including theHill reaction, the Mehler reaction and the system I reaction. (Received May 18, 1979; )     

Flux balance analysis of primary metabolism in the diatom Phaeodactylum tricornutum

Diatoms (Bacillarophyceae) are photosynthetic unicellular microalgae that have risen to ecological prominence in oceans over the past 30 million years. They are of interest as potential feedstocks for sustainable biofuels. Maximizing production of these feedstocks will require genetic modifications and an understanding of algal metabolism. These processes may benefit from genome‐scale models, which predict intracellular fluxes and theoretical yields, as well as the viability of knockout and knock‐in transformants. Here we present a genome‐scale metabolic model of a fully sequenced and transformable diatom: Phaeodactylum tricornutum. The metabolic network was constructed using the P. tricornutum genome, biochemical literature, and online bioinformatic databases. Intracellular fluxes in P. tricornutum were calculated for autotrophic, mixotrophic and heterotrophic growth conditions, as well as knockout conditions that explore the in silico role of glycolytic enzymes in the mitochondrion. The flux distribution for lower glycolysis in the mitochondrion depended on which transporters for TCA cycle metabolites were included in the model. The growth rate predictions were validated against experimental data obtained using chemostats. Two published studies on this organism were used to validate model predictions for cyclic electron flow under autotrophic conditions, and fluxes through the phosphoketolase, glycine and serine synthesis pathways under mixotrophic conditions. Several gaps in annotation were also identified. The model also explored unusual features of diatom metabolism, such as the presence of lower glycolysis pathways in the mitochondrion, as well as differences between P. tricornutum and other photosynthetic organisms.     

Identification of sequence motifs in Lhcx proteins that confer qE-based photoprotection in the diatom Phaeodactylum tricornutum

Photosynthetic organisms in nature often experience light fluctuations. While low light conditions limit the energy uptake by algae, light absorption exceeding the maximal rate of photosynthesis may go along with enhanced formation of potentially toxic reactive oxygen species. To preempt high light-induced photodamage, photosynthetic organisms evolved numerous photoprotective mechanisms. Among these, energy-dependent fluorescence quenching (qE) provides a rapid mechanism to dissipate thermally the excessively absorbed energy. Diatoms thrive in all aquatic environments and thus belong to the most important primary producers on earth. qE in diatoms is provided by a concerted action of Lhcx proteins and the xanthophyll cycle pigment diatoxanthin. While the exact Lhcx activation mechanism of diatom qE is unknown, two lumen-exposed acidic amino acids within Lhcx proteins were proposed to function as regulatory switches upon light-induced lumenal acidification. By introducing a modified Lhcx1 lacking these amino acids into a Phaeodactylum tricornutum Lhcx1-null qE knockout line, we demonstrate that qE is unaffected by these two amino acids. Based on sequence comparisons with Lhcx4, being incapable of providing qE, we perform domain swap experiments of Lhcx4 with Lhcx1 and identify two peptide motifs involved in conferring qE. Within one of these motifs, we identify a tryptophan residue with a major influence on qE establishment. This tryptophan residue is located in close proximity to the diadinoxanthin/diatoxanthin-binding site based on the recently revealed diatom Lhc crystal structure. Our findings provide a structural explanation for the intimate link of Lhcx and diatoxanthin in providing qE in diatoms.     

The diversity of small non-coding RNAs in the diatom Phaeodactylum tricornutum

Background

Marine diatoms constitute a major component of eukaryotic phytoplankton and stand at the crossroads of several evolutionary lineages. These microalgae possess peculiar genomic features and novel combinations of genes acquired from bacterial, animal and plant ancestors. Furthermore, they display both DNA methylation and gene silencing activities. Yet, the biogenesis and regulatory function of small RNAs (sRNAs) remain ill defined in diatoms.

Results

Here we report the first comprehensive characterization of the sRNA landscape and its correlation with genomic and epigenomic information in Phaeodactylum tricornutum. The majority of sRNAs is 25 to 30 nt-long and maps to repetitive and silenced Transposable Elements marked by DNA methylation. A subset of this population also targets DNA methylated protein-coding genes, suggesting that gene body methylation might be sRNA-driven in diatoms. Remarkably, 25-30 nt sRNAs display a well-defined and unprecedented 180 nt-long periodic distribution at several highly methylated regions that awaits characterization. While canonical miRNAs are not detectable, other 21-25 nt sRNAs of unknown origin are highly expressed. Besides, non-coding RNAs with well-described function, namely tRNAs and U2 snRNA, constitute a major source of 21-25 nt sRNAs and likely play important roles under stressful environmental conditions.

Conclusions

P. tricornutum has evolved diversified sRNA pathways, likely implicated in the regulation of largely still uncharacterized genetic and epigenetic processes. These results uncover an unexpected complexity of diatom sRNA population and previously unappreciated features, providing new insights into the diversification of sRNA-based processes in eukaryotes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-698) contains supplementary material, which is available to authorized users.     

The photosynthetic cytochrome c 550 from the diatom Phaeodactylum tricornutum

Photosynthesis Research - The photosynthetic cytochrome c 550 from the marine diatom Phaeodactylum tricornutum has been purified and characterized. Cytochrome c 550 is mostly obtained from the...     

Some properties of the chlorophyll fluorescence of the diatom Phaeodactylum tricornutum

Fluorescence transients were investigated with the diatom Phaeodactylumtricornutum. Supplementary experiments were done with Chaetocerossp. Under weak excitation ({small tilde}10³ erg/cm²sec), fluorescencetransients were induced simply by die oxidation-reduction reactionof Q, the primary reductant of photosystem II. The action spectraindicated that the electron transfer components between thetwo photosystems were in the most reduced state when fucoxanthinwas excited. The transients were observed with the 681 run emissionand with the 707 nm emission at room temperature. At –196°C,induction due to the reduction of Q. appeared both at the 681and 707 nm emissions. Similar results were also obtained withChaetoceros sp. Under strong excitation (10⁴–10⁵ erg/cm²-sec), the fluorescencetransients due to the interconversion between States 1 and 2of die pigment system (cf. ref. 27, 29) were observed. The transientswere induced by die alternate excitation of chlorophyll a andfucoxanthin or chlorophyll c. Conversion from State 2 to State1 was inhibited by DNP and CCCP, indicating that die processwas energy-dependent. Fluorescence spectra at –196°Cwere not altered by die state-conversion of die pigment system. These results suggest diat all die fluorescence bands whichappeared at room temperature and at –196°C were dueto die chlorophyll a of pigment system II in Phaeodactylum andChaetoceros. (Received September 7, 1972; )     

Cyclic electron transfer in photosystem II in the marine diatom Phaeodactylum tricornutum

In Phaeodactylum tricornutum Photosystem II is unusually resistant to damage by exposure to high light intensities. Not only is the capacity to dissipate excess excitations in the antenna much larger and induced more rapidly than in other organisms, but in addition an electron transfer cycle in the reaction center appears to prevent oxidative damage when secondary electron transport cannot keep up with the rate of charge separations. Such cyclic electron transfer had been inferred from oxygen measurements suggesting that some of its intermediates can be reduced in the dark and can subsequently compete with water as an electron donor to Photosystem II upon illumination. Here, the proposed activation of cyclic electron transfer by illumination is confirmed and shown to require only a second. On the other hand the dark reduction of its intermediates, specifically of tyrosine Y(D), the only Photosystem II component known to compete with water oxidation, is ruled out. It appears that the cyclic electron transfer pathway can be fully opened by reduction of the plastoquinone pool in the dark. Oxygen evolution reappears after partial oxidation of the pool by Photosystem I, but the pool itself is not involved in cyclic electron transfer.     

Cloning and characterization of a novel diacylglycerol acyltransferase from the diatom Phaeodactylum tricornutum

In this study, a cDNA encoding a novel acyl-CoA:diacylglycerol acyltransferase (DGAT)-like protein is identified and isolated from the diatom microalga Phaeodactylum tricornutum (PtDGAT3). Analysis of the sequence reveals that ptDGAT3 cDNA encodes a protein of 504 amino acids with a molecular mass of 64.5 KDa. The putative ptDGAT3 protein has two catalytic domains: a wax ester synthase-like acyl-CoA acyltransferase domain and a bacteria-specific acyltransferase domain, which shows higher similarity to the DGAT3 of Acinetobacter calcoaceticus than reported DGAT1 or DGAT2 from high plants or algae. Its activity was confirmed by heterologous expression of PtDGAT3 in a neutral lipid-deficient quadruple mutant yeast Saccharomyces cerevisiae H1246. The recombinant yeast restored the formation of a lipid body and displayed a preference to the incorporation of unsaturated C₁₈ fatty acids into triacyglycerol (TAG). This is the first characterized algal DGAT3 gene, giving further evidence to the occurrence of a DGAT3-mediated TAG biosynthesis pathway.