Intracellular distribution of the reductive and oxidative pentose phosphate pathways in two diatoms
2009, Gruber, Ansgar, Weber, Till, Río Bártulos, Carolina, Vugrinec, Sascha, Kroth, Peter G.
Diatoms contribute a large proportion to the worldwide primary production and are particularly effective in fixing carbon dioxide. Possibly because diatom plastids originate from a secondary endocytobiosis, their cellular structure is more complex and metabolic pathways are rearranged within diatom cells compared to cells containing primary plastids. We annotated genes encoding isozymes of the reductive and oxidative pentose phosphate pathways in the genomes of the centric diatom Thalassiosira pseudonana and the pennate diatom Phaeodactylum tricornutum and bioinformatically inferred their intracellular distribution. Prediction results were confirmed by fusion of selected presequences to Green Fluorescent Protein and expression of these constructs in P. tricornutum. Calvin cycle enzymes for the carbon fixation and reduction of 3-phosphoglycerate are present in single isoforms, while we found multiple isoenzymes involved in the regeneration of ribulose-1,5-bisphosphate. We only identified one cytosolic sedoheptulose-1,7-bisphosphatase in both investigated diatoms. The oxidative pentose phosphate pathway seems to be restricted to the cytosol in diatoms, since we did not find stromal glucose-6-phosphate dehydrogenase and 6-phosphogluconolactone dehydrogenase isoforms. However, the two species apparently possess a plastidic phosphogluconolactonase. A 6-phosphogluconolactone dehydrogenase is apparently plastid associated in P. tricornutum and might be active in the periplastidic compartment, suggesting that this compartment might be involved in metabolic processes in diatoms.
The Phaeodactylum genome reveals the evolutionary history of diatom genomes
2008, Bowler, Chris, Allen, Andrew E., Badger, Jonathan H., Grimwood, Jane, Jabbari, Kamel, Kuo, Alan, Maheswari, Uma, Martens, Cindy, Maumus, Florian, Otillar, Robert P., Rayko, Edda, Salamov, Asaf, Vandepoele, Klaas, Beszteri, Bank, Gruber, Ansgar, Heijde, Marc, Katinka, Michael, Mock, Thomas, Valentin, Klaus, Verret, Fréderic, Berges, John A., Brownlee, Colin, Cadoret, Jean-Paul, Chiovitti, Anthony, Choi, Chang Jae, Coesel, Sacha, De Martino, Alessandra, Detter, John Chris, Durkin, Colleen, Falciatore, Angela, Fournet, Jérome, Haruta, Miyoshi, Huysman, Marie J. J., Jenkins, Bethany D., Jiroutova, Katerina, Jorgensen, Richard E., Joubert, Yolaine, Kaplan, Aaron, Kröger, Nils, Kroth, Peter G., La Roche, Julie, Lindquist, Erica, Lommer, Markus, Martin Jézéquel, Véronique, Lopez, Pascal J., Lucas, Susan, Mangogna, Manuela, McGinnis, Karen, Medlin, Linda K., Montsant, Anton, Oudot Le Secq, Marie-Pierre, Napoli, Carolyn, Obornik, Miroslav, Schnitzler Parker, Micaela, Petit, Jean-Louis, Porcel, Betina M., Poulsen, Nicole, Robison, Matthew, Rychlewski, Leszek, Rynearson, Tatiana A., Schmutz, Jeremy, Shapiro, Harris, Siaut, Magali, Stanley, Michele S., Sussman, Michael R., Taylor, Alison R., Vardi, Assaf, Dassow, Peter von, Vyverman, Wim, Willis, Anusuya, Wyrwicz, Lucjan S., Rokhsar, Daniel S., Weissenbach, Jean, Armbrust, E. Virginia, Green, Beverley R., Van de Peer, Yves, Grigoriev, Igor V.
Diatoms are photosynthetic secondary endosymbionts found throughout marine and freshwater environments, and are believed to be responsible for around one-fifth of the primary productivity on Earth. The genome sequence of the marine centric diatom Thalassiosira pseudonana was recently reported, revealing a wealth of information about diatom biology. Here we report the complete genome sequence of the pennate diatom Phaeodactylum tricornutum and compare it with that of T. pseudonana to clarify evolutionary origins, functional significance and ubiquity of these features throughout diatoms. In spite of the fact that the pennate and centric lineages have only been diverging for 90 million years, their genome structures are dramatically different and a substantial fraction of genes (40%) are not shared by these representatives of the two lineages. Analysis of molecular divergence compared with yeasts and metazoans reveals rapid rates of gene diversification in diatoms. Contributing factors include selective gene family expansions, differential losses and gains of genes and introns, and differential mobilization of transposable elements. Most significantly, we document the presence of hundreds of genes from bacteria. More than 300 of these gene transfers are found in both diatoms, attesting to their ancient origins, and many are likely to provide novel possibilities for metabolite management and for perception of environmental signals. These findings go a long way towards explaining the incredible diversity and success of the diatoms in contemporary oceans.
Diatom plastids depend on nucleotide import from the cytosol
2009, Ast, Michelle, Gruber, Ansgar, Schmitz-Esser, Stephan, Neuhaus, Horst Ekkehard, Kroth, Peter G., Horn, Matthias, Haferkamp, Ilka
Diatoms are ecologically important algae that acquired their plastids by secondary endosymbiosis, resulting in a more complex cell structure and an altered distribution of metabolic pathways when compared with organisms with primary plastids. Diatom plastids are surrounded by 4 membranes; the outermost membrane is continuous with the endoplasmic reticulum. Genome analyses suggest that nucleotide biosynthesis is, in contrast to higher plants, not located in the plastid, but in the cytosol. As a consequence, nucleotides have to be imported into the organelle. However, the mechanism of nucleotide entry into the complex plastid is unknown. We identified a high number of putative nucleotide transporters (NTTs) in the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum and characterized the first 2 isoforms (NTT1 and NTT2). GFP-based localization studies revealed that both investigated NTTs are targeted to the plastid membranes, and that NTT1 most likely enters the innermost plastid envelope via the stroma. Heterologously expressed NTT1 acts as a proton-dependent adenine nucleotide importer, whereas NTT2 facilitates the counter exchange of (deoxy-)nucleoside triphosphates. Therefore, these transporters functionally resemble NTTs from obligate intracellular bacteria with an impaired nucleotide metabolism rather than ATP/ADP exchanging NTTs from primary plastids. We suggest that diatoms harbor a specifically-adapted nucleotide transport system and that NTTs are the key players in nucleotide supply to the complex plastid.
A Model for Carbohydrate Metabolism in the Diatom Phaeodactylum tricornutum Deduced from Comparative Whole Genome Analysis
2008, Kroth, Peter G., Chiovitti, Anthony, Gruber, Ansgar, Martin-Jezequel, Veronique, Mock, Thomas, Schnitzler Parker, Micaela, Stanley, Michele S., Kaplan, Aaron, Caron, Lise, Weber, Till, Maheswari, Uma, Armbrust, Elisabeth Virginia, Bowler, Chris, Kroymann, Juergen
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.
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.
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.
The Presence and Localization of Thioredoxins in Diatoms, Unicellular Algae of Secondary Endosymbiotic Origin
2009, Weber, Till, Gruber, Ansgar, Kroth, Peter G.
Diatoms are unicellular algae of great ecological importance. So far, very little is known about the regulation of carbon fixation in these algae; however, there are strong indications that in diatom plastids, the ferredoxin/thioredoxin system might play a minor role in redox regulation of the photosynthetic reactions compared to land plants. Until now, it is unknown whether there are fewer or other target enzymes of thioredoxins in diatoms. Only a single potential target enzyme for thioredoxin, the plastidic fructose-1,6-bisphosphatase, has yet been identified. Nevertheless, during the annotation of the genome of the diatom Phaeodactylum tricornutum, we identified several genes encoding different thioredoxins. Utilizing in vivo expression of GFP:presequence fusion proteins in P. tricornutum, we were able to show that these thioredoxins are targeted either into plastids, mitochondria, or remain in the cytosol. Surprisingly, two of the three usually cytosolic thioredoxin h proteins are apparently plastid associated and, together with a thioredoxin reductase, putatively located in the periplastidic compartment. This is one of the few indications for so far unknown enzymatic reactions in the space between the two pairs of diatom plastid envelope membranes.
Protein targeting into complex diatom plastids: functional characterization of a specific targeting motif
2007, Gruber, Ansgar, Vugrinec, Sascha, Hempel, Franziska, Gould, Sven B., Maier, Uwe-G., Kroth, Peter G.
Plastids of diatoms and related algae evolved by secondary endocytobiosis, the uptake of a eukaryotic alga into a eukaryotic host cell and its subsequent reduction into an organelle. As a result diatom plastids are surrounded by four membranes. Protein targeting of nucleus encoded plastid proteins across these membranes depends on N-terminal bipartite presequences consisting of a signal and a transit peptide-like domain. Diatoms and cryptophytes share a conserved amino acid motif of unknown function at the cleavage site of the signal peptides (ASAFAP), which is particularly important for successful plastid targeting. Screening genomic databases we found that in rare cases the very conserved phenylalanine within the motif may be replaced by tryptophan, tyrosine or leucine. To test such unusual presequences for functionality and to better understand the role of the motif and putative receptor proteins involved in targeting, we constructed presequence: GFP fusion proteins with or without modifications of the "ASAFAP"-motif and expressed them in the diatom Phaeodactylum tricornutum. In this comprehensive mutational analysis we found that only the aromatic amino acids phenylalanine, tryptophan, tyrosine and the bulky amino acid leucine at the +1 position of the predicted signal peptidase cleavage site allow plastid import, as expected from the sequence comparison of native plastid targeting presequences of P. tricornutum and the cryptophyte Guillardia theta. Deletions within the signal peptide domains also impaired plastid import, showing that the presence of F at the N-terminus of the transit peptide together with a cleavable signal peptide is crucial for plastid import.