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.     

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; )     

Changes in photosynthetic activity of the diatom Phaeodactylum tricornutum in a culture of limited volume

Changes in photosynthetic activity of a marine diatom duringalgal growth were studied with a typical culture medium formarine algae, ASP-2 (l). As the algal specimen, Phaeodactylumtricornutum was used for the experiments. Nitzschia closteriumand Chaetoceros sp. were also supplementarily used. Photosynthetic and p-benzoquinone Hill activities remarkablychanged with time during algalgrowth; with maximum activityfound in cells at log phase. A rapid decrease occurred in theinterphase from the log to stationary phase. The activity changewas not accompanied by variation in photosynthetic pigment content. The low concentration of phosphorus source was suggested asthe main cause for the change. On supply of extra inorganicphosphate, the time length for holding high photosynthetic activitybecame longer; or, the activity of the cells at stationary phaserecovered at least partly even in the dark. Dark recovery wasnot accompanied by either algal growth or an increase in thecontent of photosynthetic pigments. Inactivation of photosynthesis in the stationary growth phaseand activation by added phosphate in the dark were inferredto be due to changes in activities of both the CO₂-fixing andelectron transfer systems. The observed activity change maynot be attributable to a deficiency in inorganic phosphate asthe substrate for photophosphorylation. Similar changes in photosynthetic activity were also observedwith Nitzschia closterium and Chaetoceros sp. (Received January 30, 1971; )     

Changes in the photosynthetic reaction centre II in the diatom Phaeodactylum tricornutum result in non-photochemical fluorescence quenching

Diatoms are an important group of primary producers in the aquatic environment. They are able to acclimate to fast changes in the light intensity by various mechanisms including a rise in non-photochemical fluorescence quenching (NPQ). The latter has been attributed to the xanthophyll cycle (XC) following activation of diadinoxanthin de-epoxidase by the acidification of the thylakoid lumen. To examine whether fluorescence quenching in the diatom Phaeodactylum tricornutum depends on the ΔpH generated by the photosynthetic electron transport, we arrested the latter by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU). This treatment hardly affected the NPQ or XC, even when methylviologen was present. Dissipation of the ΔpH by 2,4-dinitrophenol inhibited the XC but did not alter NPQ. Similar results, i.e. inhibition of the XC but normal fluorescence quenching, were observed when the experiments were performed at 3°C. Measurements of thermoluminescence showed that excess light treatment caused a marked decline in the signals obtained as a result of recombination of Q_B^- with the S₃ state of the Mn cluster; this was also observed in cells treated with DCMU (recombination of Q_A^- with S₂). Light treatment also diminished the Q_A^- re-oxidation signals. The data suggest that changes in PSII core centre itself due to exposure to excess light conditions play an important part in the acclimation of P. tricornutum to the changing light conditions.     

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.     

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。     

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; )     

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.     

Response of the diatom Phaeodactylum tricornutum to photooxidative stress resulting from high light exposure

The response of microalgae to photooxidative stress resulting from high light exposure is a well-studied phenomenon. However, direct analyses of photosystem II (PSII) D1 protein (the main target of photoinhibition) in diatoms are scarce. In this study, the response of the diatom model species Phaeodactylum tricornutum to short-term exposure to high light was examined and the levels of D1 protein determined immunochemically. Low light (LL) acclimated cells (40 μmol photons m(-2) s(-1)) subjected to high light (HL, 1,250 μmol photons m(-2) s(-1)) showed rapid induction of non-photochemical quenching (NPQ) and ca. 20-fold increase in diatoxanthin (DT) concentration. This resulted from the conversion of diadinoxanthin (DD) to DT through the activation of the DD-cycle. D1 protein levels under LL decreased about 30% after 1 h of the addition of lincomycin (LINC), a chloroplast protein synthesis inhibitor, showing significant D1 degradation and repair under low irradiance. Exposure to HL lead to a 3.2-fold increase in D1 degradation rate, whereas average D1 repair rate was 1.3-x higher under HL than LL, leading to decreased levels of D1 protein under HL. There were significant effects of both HL and LINC on P. tricornutum maximum quantum yield of PSII (F(v)/F(m)), showing a reduction of active PSII reaction centres. Partial recovery of F(v)/F(m) in the dark demonstrates the photosynthetic resilience of this diatom to changes in the light regime. P. tricornutum showed high allocation of total protein to D1 and an active D1-repair cycle to limit photoinhibition.     

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.     

Research note: High efficiency transformation of the diatom Phaeodactylum tricornutum with a promoter from the diatom Cylindrotheca fusiformis

A high efficiency transformation system was established for the pennate diatom Phaeodactylum tricornutum Bohlin using a plasmid containing fucoxanthin chlorophyll a/c binding protein ( fcp ) promoter/terminator and nitrate reductase ( NR ) promoter/terminator that are derived from the pennate diatom Cylindrotheca fusiformis . The plasmid that contains the zeocin resistance gene ( ble ) with the fcp promoter and enhanced green fluorescent protein gene ( egfp ) with the NR promoter was introduced into P. tricornutum using microparticle bombardment. Transformants (650 ± 58 per 10⁸ cells) were obtained. The yield of transformants was between 1.5 and 130 times higher than previously reported P. tricornutum transformation systems. Four to seven copies of the ble gene were integrated into genomic DNA of the transformants. This high efficiency transformation system of P. tricornutum is expected to provide a powerful tool for high-throughput analysis of gene function using homologous recombination or RNAi.     

The light-harvesting antenna of the diatom Phaeodactylum tricornutum. Evidence for a diadinoxanthin-binding subcomplex

Diatoms differ from higher plants by their antenna system, in terms of both polypeptide and pigment contents. A rapid isolation procedure was designed for the membrane-intrinsic light harvesting complexes (LHC) of the diatom Phaeodactylum tricornutum to establish whether different LHC subcomplexes exist, as well to determine an uneven distribution between them of pigments and polypeptides. Two distinct fractions were separated that contain functional oligomeric complexes. The major and more stable complex ( approximately 75% of total polypeptides) carries most of the chlorophyll a, and almost only one type of carotenoid, fucoxanthin. The minor complex, carrying approximately 10-15% of the total antenna chlorophyll and only a little chlorophyll c, is highly enriched in diadinoxanthin, the main xanthophyll cycle carotenoid. The two complexes also differ in their polypeptide composition, suggesting specialized functions within the antenna. The diadinoxanthin-enriched complex could be where the de-epoxidation of diadinoxanthin into diatoxanthin mostly occurs.     

Physiological and molecular biological characterization of intracellular carbonic anhydrase from the marine diatom Phaeodactylum tricornutum

A single intracellular carbonic anhydrase (CA) was detected in air-grown and, at reduced levels, in high CO(2)-grown cells of the marine diatom Phaeodactylum tricornutum (UTEX 642). No external CA activity was detected irrespective of growth CO(2) conditions. Ethoxyzolamide (0.4 mM), a CA-specific inhibitor, severely inhibited high-affinity photosynthesis at low concentrations of dissolved inorganic carbon, whereas 2 mM acetazolamide had little effect on the affinity for dissolved inorganic carbon, suggesting that internal CA is crucial for the operation of a carbon concentrating mechanism in P. tricornutum. Internal CA was purified 36.7-fold of that of cell homogenates by ammonium sulfate precipitation, and two-step column chromatography on diethylaminoethyl-sephacel and p-aminomethylbenzene sulfone amide agarose. The purified CA was shown, by SDS-PAGE, to comprise an electrophoretically single polypeptide of 28 kD under both reduced and nonreduced conditions. The entire sequence of the cDNA of this CA was obtained by the rapid amplification of cDNA ends method and indicated that the cDNA encodes 282 amino acids. Comparison of this putative precursor sequence with the N-terminal amino acid sequence of the purified CA indicated that it included a possible signal sequence of up to 46 amino acids at the N terminus. The mature CA was found to consist of 236 amino acids and the sequence was homologous to beta-type CAs. Even though the zinc-ligand amino acid residues were shown to be completely conserved, the amino acid residues that may constitute a CO(2)-binding site appeared to be unique among the beta-CAs so far reported.     

Spectroscopic and molecular characterization of the oligomeric antenna of the diatom Phaeodactylum tricornutum

The photosynthetic antenna system of diatoms contains fucoxanthin chlorophyll a/c binding proteins (FCPs), which are membrane intrinsic proteins showing high homology to the light harvesting complexes (LHC) of higher plants. In the present study, we used a mild solubilization of P. tricornutum thylakoid membranes in combination with sucrose density gradient centrifugation or gelfiltration and obtained an oligomeric FCP complex (FCPo). The spectroscopic characteristics and pigment stoichiometries of the FCPo complex were comparable to FCP complexes that were isolated after solubilization with higher detergent per chlorophyll ratios. The excitation energy transfer between the FCP-bound pigments was more efficient in the oligomeric FCPo complexes, indicating that these complexes may represent the native form of the diatom antenna system in the thylakoid membrane. Determination of the molecular masses of the two different FCP fractions by gelfiltration revealed that the FCP complexes consisted of trimers, whereas the FCPo complexes were either composed of six monomers or two tightly associated trimers. In contrast to vascular plants, stable functional monomers could not be isolated in P. tricornutum. Both types of FCP complexes showed two protein bands in SDS-gels with apparent molecular masses of 18 and 19 kDa, respectively. Sequence analysis by MS/MS revealed that the 19 kDa protein corresponded to the fcpC and fcpD genes, whereas the 18 kDa band contained the protein of the fcpE gene. The presence of an oligomeric antenna in diatoms is in line with the oligomeric organization of antenna complexes in different photoautotrophic groups.