The Diatom EST database provides integrated access to expressed sequence tag (EST) data from two ... more The Diatom EST database provides integrated access to expressed sequence tag (EST) data from two eukaryotic microalgae of the class Bacillariophyceae, Phaeodactylum tricornutum and Thalassiosira pseudonana. The database currently contains sequences of close to 30 000 ESTs organized into PtDB, the P.tricornutum EST database, and TpDB, the T.pseudonana EST database. The EST sequences were clustered and assembled into a non-redundant set for each organism, and these non-redundant sequences were then subjected to automated annotation using similarity searches against protein and domain databases. EST sequences, clusters of contiguous sequences, their annotation and analysis with reference to the publicly available databases, and a codon usage table derived from a subset of sequences from PtDB and TpDB can all be accessed in the Diatom EST Database. The underlying RDBMS enables queries over the raw and annotated EST data and retrieval of information through a user-friendly
To obtain a copy of this Journal Article, link to the DOI if present for specific Publisher infor... more To obtain a copy of this Journal Article, link to the DOI if present for specific Publisher information. Library patrons may click here to search WorldCat to identify libraries that hold this journal title; check beforehand to verify the library holds the volume needed. ... Select a citation type above to copy/paste or download the reference. ... Some links on this page may take you to non-federal websites. Their policies may differ from this site.
Aim of study: To assess the impacts of climate change on local agriculture with a high resolution... more Aim of study: To assess the impacts of climate change on local agriculture with a high resolution in a Mediterranean region with a diversity of climates.Area of study: Catalonia (NW Spain).Material and methods: Based on historical meteorological records and a regionalization of the RCP4.5 model created by the Catalan Meteorological Service, the Papadakis agro-climate classification was calculated for two climate scenarios. The changes in agro-climatic suitability and irrigation needs of five typical Mediterranean crops (alfalfa, almond, barley, olive and orange) were analysed. Main results: In the 2031-2050 climate scenario, over 15% of the study area will no longer be adequate for non-irrigated almond or olive, at locations in which they have been traditionally rainfed crops. If irrigation is provided, orange is likely to become agro-climatically suited for the entire Catalan coastline. Were the current crop distribution maintained, irrigation needs may increase on average 16% in t...
Diatoms are one of the most important constituents of phytoplankton communities in aquatic enviro... more 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. ; fax 33-1-44-32-39-35.
The Diatom EST database provides integrated access to expressed sequence tag (EST) data from two ... more The Diatom EST database provides integrated access to expressed sequence tag (EST) data from two eukaryotic microalgae of the class Bacillariophyceae, Phaeodactylum tricornutum and Thalassiosira pseudonana. The database currently contains sequences of close to 30 000 ESTs organized into PtDB, the P.tricornutum EST database, and TpDB, the T.pseudonana EST database. The EST sequences were clustered and assembled into a non-redundant set for each organism, and these non-redundant sequences were then subjected to automated annotation using similarity searches against protein and domain databases. EST sequences, clusters of contiguous sequences, their annotation and analysis with reference to the publicly available databases, and a codon usage table derived from a subset of sequences from PtDB and TpDB can all be accessed in the Diatom EST Database. The underlying RDBMS enables queries over the raw and annotated EST data and retrieval of information through a user-friendly web interface, with options to perform keyword and BLAST searches. The EST data can also be retrieved based on Pfam domains, Cluster of Orthologous Groups (COG) and Gene Ontologies (GO) assigned to them by similarity searches. The Database is available at http://avesthagen.sznbowler.com.
Diatoms are photosynthetic secondary endosymbionts found throughout marine and freshwater environ... more 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 1,2 . The genome sequence of the marine centric diatom Thalassiosira pseudonana was recently reported, revealing a wealth of information about diatom biology 3-5 . 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.
Diatoms and other chlorophyll-c containing, or chromalveolate, algae are among the most productiv... more Diatoms and other chlorophyll-c containing, or chromalveolate, algae are among the most productive and diverse phytoplankton in the ocean. Evolutionarily, chlorophyll-c algae are linked through common, although not necessarily monophyletic, acquisition of plastid endosymbionts of red as well as most likely green algal origin. There is also strong evidence for a relatively high level of lineage-specific bacterial gene acquisition within chromalveolates. Therefore, analyses of gene content and derivation in chromalveolate taxa have indicated particularly diverse origins of their overall gene repertoire. As a single group of functionally related enzymes spanning two distinct gene families, fructose 1,6-bisphosphate aldolases (FBAs) illustrate the influence on core biochemical pathways of specific evolutionary associations among diatoms and other chromalveolates with various plastid-bearing and bacterial endosymbionts. Protein localization and activity, gene expression, and phylogenetic analyses indicate that the pennate diatom Phaeodactylum tricornutum contains five FBA genes with very little overall functional overlap. Three P. tricornutum FBAs, one class I and two class II, are plastid localized, and each appears to have a distinct evolutionary origin as well as function. Class I plastid FBA appears to have been acquired by chromalveolates from a red algal endosymbiont, whereas one copy of class II plastid FBA is likely to have originated from an ancient green algal endosymbiont. The other copy appears to be the result of a chromalveolate-specific gene duplication. Plastid FBA I and chromalveolate-specific class II plastid FBA are localized in the pyrenoid region of the chloroplast where they are associated with b-carbonic anhydrase, which is known to play a significant role in regulation of the diatom carbon concentrating mechanism. The two pyrenoid-associated FBAs are distinguished by contrasting gene expression profiles under nutrient limiting compared with optimal CO 2 fixation conditions, suggestive of a distinct specialized function for each. Cytosolically localized FBAs in P. tricornutum likely play a role in glycolysis and cytoskeleton function and seem to have originated from the stramenopile host cell and from diatom-specific bacterial gene transfer, respectively.
The green alga Haematococcus pluvialis accumulates massive amounts of the red pigment astaxanthin... more The green alga Haematococcus pluvialis accumulates massive amounts of the red pigment astaxanthin in response to stimuli inducing it to form cysts. During the encystment process the cell wall undergoes a clear hardening and thickening. In this work, a cell wall fraction withstanding successive acid and basic hydrolysis was isolated and proves to be algaenan by Fourier transform infrared spectroscopy. This compound is equally abundant in nonmotile vegetative cells and astaxanthin-rich cysts. This finding indicates that the synthesis of algaenan does not require the activation of the machinery for the massive production of secondary carotenoids. We conclude that algaenan cannot cause the changes occurring in the cell wall during the encystment process without the involvement of other cell wall components.
For diatom biologists one of the most interesting research areas over the next years will be in l... more For diatom biologists one of the most interesting research areas over the next years will be in linking mathematical models for pattern formation with information derived from molecular genetic, biochemical, and physiological studies. A major goal of this research is to exploit diatom proficiency in biogenic silica formation to develop strategies for bio-inspired nanofabrication of silicon based materials. Development of high-throughput methods for the functional analysis of diatom genes is a key step toward this goal. In this article we review the different techniques available to investigate gene and protein function in diatoms. Furthermore, to make diatom research as effective as possible the research community must address the question of which diatom species should be developed as a model. Choice of a diatom model organism should be made on the basis of several criteria, such as the ease of genetic manipulation, ecological relevance, or biomineralization capability. Phaeodactylum tricornutum is one of the principal three species that are candidates for such a model. For this species we have accomplished the first large-scale analysis of 12 000 expressed sequence tags (ESTs) and have organized it in a queryable database, Phaeodactylum tricornutum database (PtDB). A summary of the functional analysis of this EST collection is presented, and genes of particular interest are highlighted.
Research into diatom biology has now entered the post-genomics era, following the recent completi... more 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.
To obtain a copy of this Journal Article, link to the DOI if present for specific Publisher infor... more To obtain a copy of this Journal Article, link to the DOI if present for specific Publisher information. Library patrons may click here to search WorldCat to identify libraries that hold this journal title; check beforehand to verify the library holds the volume needed. ... Select a citation type above to copy/paste or download the reference. ... Some links on this page may take you to non-federal websites. Their policies may differ from this site.
The marine pennate diatom Phaeodactylum tricornutum has become a model for diatom biology, due to... more The marine pennate diatom Phaeodactylum tricornutum has become a model for diatom biology, due to its ease of culture and accessibility to reverse genetics approaches. While several features underlying the molecular mechanisms of cell division have been described, morphological analyses are less advanced than they are in other diatoms. We therefore examined cell ultrastructure changes prior to and during cytokinesis. Following chloroplast division, cleavage furrows are formed at both longitudinal ends of the cell and are accompanied by significant vesicle transport. Although neither spindle nor microtubules were observed, the nucleus appeared to be split by the furrow after duplication of the Golgi apparatus. Finally, centripetal cytokinesis was completed by fusion of the furrows. Additionally, F-actin formed a ring structure and its diameter became smaller, accompanying the ingrowing furrows. To further analyse vesicular transport during cytokinesis, we generated transgenic cells expressing yellow fluorescent protein (YFP) fusions with putative diatom orthologs of small GTPase Sec4 and t-SNARE protein SyntaxinA. Time-lapse observations revealed that SyntaxinA-YFP localization expands from both cell tips toward the center, whereas Sec4-YFP was found in the Golgi and subsequently relocalizes to the future division plane. This work provides fundamental new information about cell replication processes in P. tricornutum. W (2005) Membrane traffic: a driving force in cytokinesis. Trends Cell Biol 15:92-101 Allen AE, Dupont CL, Oborník M, Horák A, Nunes-Nesi A, McCrow JP, Zheng H, Johnson DA, Hu H, Fernie AR, Bowler C (2011) Evolution and metabolic significance of the urea cycle in photosynthetic diatoms. Nature 473:203-207 Armbrust EV, et al. (2004) The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 306:79-86 Assaad FF, Huet Y, Mayer U, Jürgens G (2001) The cytokinesis gene KEULE encodes a Sac1 protein that binds the syntaxin KNOLLE. J Cell Biol 152:531-543 Barr FA, Gruneberg U (2007) Cytokinesis: placing and making the final cut. Cell 131:847-860 Bennett MK, Calakos N, Scheller RH (1992) Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science 257:255-259 Borowitzka MA, Volcani BE (1978) The polymorphic diatom Phaeodactylum tricornutum: Ultrastructure of its morphotypes. J Phycol 14:10-21 Boutté Y, Frescatada-Rosa M, Men S, Chow CM, Ebine K, Gustavsson A, Johansson L, Ueda T, Moore I, Jürgens G, Grebe M (2010) Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis. EMBO J 29:546-558 Bowler C, et al. (2008) The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 456:239-244 Bowler C, De Martino A, Falciatore A (2010) Diatom cell division in an environmental context. Curr Opin Plant Biol 13:623-630 Brzezinski MA, Olson RJ, Chisholm SW (1990) Silicon availability and cell-cycle progression in marine diatoms. Marine Ecol Prog Ser 67:83-96 Burgess RW, Deitcher DL, Schwarz TL (1997) The synaptic protein syntaxin1 is required for cellularization of Drosophila embryos. J Cell Biol 138:861-875 Cavalier-Smith T (1999) Principles of protein and lipid targeting in secondary symbiogenesis: Euglenoid, Dinoflagellate, and Sporozoan plastid origins and the eukaryote family tree. J Eukaryot Microbiol 46:347-366 Cohn SA, Nash J, Picket-Heaps JD (1989) The effect of drugs on diatom valve morphogenesis. Protoplasma
The Diatom EST database provides integrated access to expressed sequence tag (EST) data from two ... more The Diatom EST database provides integrated access to expressed sequence tag (EST) data from two eukaryotic microalgae of the class Bacillariophyceae, Phaeodactylum tricornutum and Thalassiosira pseudonana. The database currently contains sequences of close to 30 000 ESTs organized into PtDB, the P.tricornutum EST database, and TpDB, the T.pseudonana EST database. The EST sequences were clustered and assembled into a non-redundant set for each organism, and these non-redundant sequences were then subjected to automated annotation using similarity searches against protein and domain databases. EST sequences, clusters of contiguous sequences, their annotation and analysis with reference to the publicly available databases, and a codon usage table derived from a subset of sequences from PtDB and TpDB can all be accessed in the Diatom EST Database. The underlying RDBMS enables queries over the raw and annotated EST data and retrieval of information through a user-friendly
To obtain a copy of this Journal Article, link to the DOI if present for specific Publisher infor... more To obtain a copy of this Journal Article, link to the DOI if present for specific Publisher information. Library patrons may click here to search WorldCat to identify libraries that hold this journal title; check beforehand to verify the library holds the volume needed. ... Select a citation type above to copy/paste or download the reference. ... Some links on this page may take you to non-federal websites. Their policies may differ from this site.
Aim of study: To assess the impacts of climate change on local agriculture with a high resolution... more Aim of study: To assess the impacts of climate change on local agriculture with a high resolution in a Mediterranean region with a diversity of climates.Area of study: Catalonia (NW Spain).Material and methods: Based on historical meteorological records and a regionalization of the RCP4.5 model created by the Catalan Meteorological Service, the Papadakis agro-climate classification was calculated for two climate scenarios. The changes in agro-climatic suitability and irrigation needs of five typical Mediterranean crops (alfalfa, almond, barley, olive and orange) were analysed. Main results: In the 2031-2050 climate scenario, over 15% of the study area will no longer be adequate for non-irrigated almond or olive, at locations in which they have been traditionally rainfed crops. If irrigation is provided, orange is likely to become agro-climatically suited for the entire Catalan coastline. Were the current crop distribution maintained, irrigation needs may increase on average 16% in t...
Diatoms are one of the most important constituents of phytoplankton communities in aquatic enviro... more 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. ; fax 33-1-44-32-39-35.
The Diatom EST database provides integrated access to expressed sequence tag (EST) data from two ... more The Diatom EST database provides integrated access to expressed sequence tag (EST) data from two eukaryotic microalgae of the class Bacillariophyceae, Phaeodactylum tricornutum and Thalassiosira pseudonana. The database currently contains sequences of close to 30 000 ESTs organized into PtDB, the P.tricornutum EST database, and TpDB, the T.pseudonana EST database. The EST sequences were clustered and assembled into a non-redundant set for each organism, and these non-redundant sequences were then subjected to automated annotation using similarity searches against protein and domain databases. EST sequences, clusters of contiguous sequences, their annotation and analysis with reference to the publicly available databases, and a codon usage table derived from a subset of sequences from PtDB and TpDB can all be accessed in the Diatom EST Database. The underlying RDBMS enables queries over the raw and annotated EST data and retrieval of information through a user-friendly web interface, with options to perform keyword and BLAST searches. The EST data can also be retrieved based on Pfam domains, Cluster of Orthologous Groups (COG) and Gene Ontologies (GO) assigned to them by similarity searches. The Database is available at http://avesthagen.sznbowler.com.
Diatoms are photosynthetic secondary endosymbionts found throughout marine and freshwater environ... more 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 1,2 . The genome sequence of the marine centric diatom Thalassiosira pseudonana was recently reported, revealing a wealth of information about diatom biology 3-5 . 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.
Diatoms and other chlorophyll-c containing, or chromalveolate, algae are among the most productiv... more Diatoms and other chlorophyll-c containing, or chromalveolate, algae are among the most productive and diverse phytoplankton in the ocean. Evolutionarily, chlorophyll-c algae are linked through common, although not necessarily monophyletic, acquisition of plastid endosymbionts of red as well as most likely green algal origin. There is also strong evidence for a relatively high level of lineage-specific bacterial gene acquisition within chromalveolates. Therefore, analyses of gene content and derivation in chromalveolate taxa have indicated particularly diverse origins of their overall gene repertoire. As a single group of functionally related enzymes spanning two distinct gene families, fructose 1,6-bisphosphate aldolases (FBAs) illustrate the influence on core biochemical pathways of specific evolutionary associations among diatoms and other chromalveolates with various plastid-bearing and bacterial endosymbionts. Protein localization and activity, gene expression, and phylogenetic analyses indicate that the pennate diatom Phaeodactylum tricornutum contains five FBA genes with very little overall functional overlap. Three P. tricornutum FBAs, one class I and two class II, are plastid localized, and each appears to have a distinct evolutionary origin as well as function. Class I plastid FBA appears to have been acquired by chromalveolates from a red algal endosymbiont, whereas one copy of class II plastid FBA is likely to have originated from an ancient green algal endosymbiont. The other copy appears to be the result of a chromalveolate-specific gene duplication. Plastid FBA I and chromalveolate-specific class II plastid FBA are localized in the pyrenoid region of the chloroplast where they are associated with b-carbonic anhydrase, which is known to play a significant role in regulation of the diatom carbon concentrating mechanism. The two pyrenoid-associated FBAs are distinguished by contrasting gene expression profiles under nutrient limiting compared with optimal CO 2 fixation conditions, suggestive of a distinct specialized function for each. Cytosolically localized FBAs in P. tricornutum likely play a role in glycolysis and cytoskeleton function and seem to have originated from the stramenopile host cell and from diatom-specific bacterial gene transfer, respectively.
The green alga Haematococcus pluvialis accumulates massive amounts of the red pigment astaxanthin... more The green alga Haematococcus pluvialis accumulates massive amounts of the red pigment astaxanthin in response to stimuli inducing it to form cysts. During the encystment process the cell wall undergoes a clear hardening and thickening. In this work, a cell wall fraction withstanding successive acid and basic hydrolysis was isolated and proves to be algaenan by Fourier transform infrared spectroscopy. This compound is equally abundant in nonmotile vegetative cells and astaxanthin-rich cysts. This finding indicates that the synthesis of algaenan does not require the activation of the machinery for the massive production of secondary carotenoids. We conclude that algaenan cannot cause the changes occurring in the cell wall during the encystment process without the involvement of other cell wall components.
For diatom biologists one of the most interesting research areas over the next years will be in l... more For diatom biologists one of the most interesting research areas over the next years will be in linking mathematical models for pattern formation with information derived from molecular genetic, biochemical, and physiological studies. A major goal of this research is to exploit diatom proficiency in biogenic silica formation to develop strategies for bio-inspired nanofabrication of silicon based materials. Development of high-throughput methods for the functional analysis of diatom genes is a key step toward this goal. In this article we review the different techniques available to investigate gene and protein function in diatoms. Furthermore, to make diatom research as effective as possible the research community must address the question of which diatom species should be developed as a model. Choice of a diatom model organism should be made on the basis of several criteria, such as the ease of genetic manipulation, ecological relevance, or biomineralization capability. Phaeodactylum tricornutum is one of the principal three species that are candidates for such a model. For this species we have accomplished the first large-scale analysis of 12 000 expressed sequence tags (ESTs) and have organized it in a queryable database, Phaeodactylum tricornutum database (PtDB). A summary of the functional analysis of this EST collection is presented, and genes of particular interest are highlighted.
Research into diatom biology has now entered the post-genomics era, following the recent completi... more 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.
To obtain a copy of this Journal Article, link to the DOI if present for specific Publisher infor... more To obtain a copy of this Journal Article, link to the DOI if present for specific Publisher information. Library patrons may click here to search WorldCat to identify libraries that hold this journal title; check beforehand to verify the library holds the volume needed. ... Select a citation type above to copy/paste or download the reference. ... Some links on this page may take you to non-federal websites. Their policies may differ from this site.
The marine pennate diatom Phaeodactylum tricornutum has become a model for diatom biology, due to... more The marine pennate diatom Phaeodactylum tricornutum has become a model for diatom biology, due to its ease of culture and accessibility to reverse genetics approaches. While several features underlying the molecular mechanisms of cell division have been described, morphological analyses are less advanced than they are in other diatoms. We therefore examined cell ultrastructure changes prior to and during cytokinesis. Following chloroplast division, cleavage furrows are formed at both longitudinal ends of the cell and are accompanied by significant vesicle transport. Although neither spindle nor microtubules were observed, the nucleus appeared to be split by the furrow after duplication of the Golgi apparatus. Finally, centripetal cytokinesis was completed by fusion of the furrows. Additionally, F-actin formed a ring structure and its diameter became smaller, accompanying the ingrowing furrows. To further analyse vesicular transport during cytokinesis, we generated transgenic cells expressing yellow fluorescent protein (YFP) fusions with putative diatom orthologs of small GTPase Sec4 and t-SNARE protein SyntaxinA. Time-lapse observations revealed that SyntaxinA-YFP localization expands from both cell tips toward the center, whereas Sec4-YFP was found in the Golgi and subsequently relocalizes to the future division plane. This work provides fundamental new information about cell replication processes in P. tricornutum. W (2005) Membrane traffic: a driving force in cytokinesis. Trends Cell Biol 15:92-101 Allen AE, Dupont CL, Oborník M, Horák A, Nunes-Nesi A, McCrow JP, Zheng H, Johnson DA, Hu H, Fernie AR, Bowler C (2011) Evolution and metabolic significance of the urea cycle in photosynthetic diatoms. Nature 473:203-207 Armbrust EV, et al. (2004) The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 306:79-86 Assaad FF, Huet Y, Mayer U, Jürgens G (2001) The cytokinesis gene KEULE encodes a Sac1 protein that binds the syntaxin KNOLLE. J Cell Biol 152:531-543 Barr FA, Gruneberg U (2007) Cytokinesis: placing and making the final cut. Cell 131:847-860 Bennett MK, Calakos N, Scheller RH (1992) Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science 257:255-259 Borowitzka MA, Volcani BE (1978) The polymorphic diatom Phaeodactylum tricornutum: Ultrastructure of its morphotypes. J Phycol 14:10-21 Boutté Y, Frescatada-Rosa M, Men S, Chow CM, Ebine K, Gustavsson A, Johansson L, Ueda T, Moore I, Jürgens G, Grebe M (2010) Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis. EMBO J 29:546-558 Bowler C, et al. (2008) The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 456:239-244 Bowler C, De Martino A, Falciatore A (2010) Diatom cell division in an environmental context. Curr Opin Plant Biol 13:623-630 Brzezinski MA, Olson RJ, Chisholm SW (1990) Silicon availability and cell-cycle progression in marine diatoms. Marine Ecol Prog Ser 67:83-96 Burgess RW, Deitcher DL, Schwarz TL (1997) The synaptic protein syntaxin1 is required for cellularization of Drosophila embryos. J Cell Biol 138:861-875 Cavalier-Smith T (1999) Principles of protein and lipid targeting in secondary symbiogenesis: Euglenoid, Dinoflagellate, and Sporozoan plastid origins and the eukaryote family tree. J Eukaryot Microbiol 46:347-366 Cohn SA, Nash J, Picket-Heaps JD (1989) The effect of drugs on diatom valve morphogenesis. Protoplasma
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