Gruber, Ansgar


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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.

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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.


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.

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Sensitivity of Laminariales zoospores from Helgoland (North Sea) to ultraviolet and photosynthetically active radiation: implications for depth distribution and seasonal reproduction

2005, Roleda, Michael Y., Wiencke, Christian, Hanelt, Dieter, van de Poll, Willem H., Gruber, Ansgar

Depth distribution of kelp species in Helgoland (North Sea) is characterized by occurrence of Laminaria digitata in the upper sublittoral, whereas L. saccharina and L. hyperborea dominate the mid and lower sublittoral region. Laminaria digitata is fertile in summer whereas both other species are fertile in autumn/winter. To determine the light sensitivity of the propagules, zoospores of L. digitata, L. saccharina and L. hyperborea were exposed in the laboratory to different exposure times of photosynthetically active radiation (PAR; 400 700 nm), PAR + UVA radiation (UVAR; 320 400 nm) and PAR + UVAR + UVB radiation (UVBR; 280 320 nm). Optimum quantum yield of PSII and DNA damage were measured after exposure. Subsequently, recovery of photosynthetic efficiency and DNA damage repair, as well as germination rate were measured after 2 and 3 d cultivation in dim white light. Photosynthetic efficiency of all species was photoinhibited already at 20 µmol photons m−2 s−1 PAR, whereas UV radiation (UVR) had a significant additional effect on photoinhibition. Recovery of the PSII function was observed in all species but not in spores exposed to irradiation longer than 4 h of PAR + UVA + UVB and 8 h of PAR + UVA. The amount of UVB-induced DNA damage measured as cyclobutane pyrimidine dimers (CPDs) increased with exposure time and highest damage was detected in the spores of lower subtidal L. hyperborea relative to the other two species. Significant removal of CPDs indicating repair of DNA damage was observed in all species after 2 d in low white light especially in the spores of upper subtidal L. digitata. Therefore, efficient DNA damage repair and recovery of PSII damage contributed to the germination success but not in spores exposed to 16 h of UVBR. UV absorption of zoospore suspension in L. digitata is based both on the absorption by the zoospores itself as well as by exudates in the medium. In contrast, the absorption of the zoospore suspension in L. saccharina and L. hyperborea is based predominantly on the absorption by the exudates in the medium. This study indicates that UVR sensitivity of zoospores is related to the seasonal zoospore production as well as the vertical distribution pattern of the large sporophytes.

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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.


Molecular Characterisation of Diatom Plastids

2008, Gruber, Ansgar

Diatoms are photoautotrophic, unicellular organisms, found in many aquatic habitats. Diatom plastids most likely evolved by secondary endocytobiosis, the uptake of a eukaryotic alga into another eukaryotic host cell and the subsequent evolutionary reduction and specialisation of this endosymbiont to a cell organelle. Diatom plastids differ from plastids of higher plants in many characteristics, e.g. they are surrounded by four (instead of two) membranes.
Plastids and mitochondria contain independent genomes that trace back to the genomes of their free living ancestors, cyanobacteria and Alphaproteobacteria, respectively. The ratio of plastid and mitochondrial genome copies to nuclear genome copies was determined via quantitative real-time polymerase chain reaction (qPCR). Fusion of a plastid targeted recombinase (RecA) to the green fluorescent protein (GFP) leads to selective labelling of plastid nucleoids in the diatom Phaeodactylum tricornutum.
Many essential photosynthesis enzymes are encoded in the nuclear genome, the gene products thus have to be imported into the plastids. A conserved sequence motif of unknown function ( ASAFAP -motif) within the N-terminal presequence of plastid preproteins is particularly important for the import reaction. The molecular characterisation of the conserved presequence motif allows conclusions on the targeting mechanisms involved and facilitates the prediction of plastid localised proteins on a genomic scale.
The genomes of the model diatoms P. tricornutum and Thalassiosira pseudonana have been sequenced completely. By analysis of the putative intracellular localisations of enzymes based on identified presequences, indications for a C4-like photosynthesis in P. tricornutum were found, and models for carbon concentrating mechanisms and CO2 fixation in P. tricornutum and T. pseudonana have been inferred. Peculiarly, multiple isoforms of enzymes of the Calvin cycle and glycolysis are present in the investigated diatoms. The isoforms differ in their presequences and are putatively active in the plastids, the mitochondria and the cytosol.
The exchange of metabolites between stroma, cytosol and other organelles is crucial for plastid function. Eight and six putative plastidic nucleotide transporters are encoded in the genomes of T. pseudonana and P. tricornutum respectively. Fusion proteins of nucleotide transporter presequences or full length fusions to GFP show that the investigated nucleotide transporters are plastid associated.


Der1-mediated Preprotein Import into the Periplastid Compartment of Chromalveolates?

2007, Sommer, Maik Sascha, Gould, Sven B., Lehmann, Petra, Gruber, Ansgar, Przyborski, Jude M., Maier, Uwe-G.

Phototrophic chromalveolates possess plastids surrounded by either 3 or 4 membranes, revealing their secondary endosymbiotic origin from an engulfed eukaryotic alga. In cryptophytes, a member of the chromalveolates, the organelle is embedded within a designated region of the host's rough endoplasmic reticulum (RER). Its eukaryotic compartments other than the plastid were reduced to the mere remains of its former cytosol, the periplastid compartment (PPC, PP space), and its nucleus, the nucleomorph, separated from the RER by its former plasma membrane, the periplast membrane (PPM). In the nucleomorph genome of the cryptophyte Guillardia theta, we identified several genes sharing homology with components of the ER-associated degradation (ERAD) machinery of yeast and higher eukaryotes, namely ORF201 and ORF477, homologs of membrane-bound proteins, Der1p (Degradation in the ER protein 1) and the RING-finger ubiquitin ligase Hrd1, and a truncated version of Udf1, a cofactor of Cdc48, a lumenal ATPase. Exemplarily, studies on the Der1-homolog ORF201 showed that this protein partially rescued a yeast deletion mutant, indicating the existence of a functional PPC-specific ERAD-like system in cryptophytes. With the noninvestigated exception of haptophytes a phylogenetically and mechanistically related system is apparently present in all chromalveolates with 4 membrane bound plastids because amongst others, PPC-specific Derlins (Der1-like proteins), CDC48 and its cofactor Ufd1 were identified in the nuclear genomes of diatoms and apicomplexa. These proteins are equipped with the required topogenic signals to direct them into the periplastid compartment of their secondary symbionts. Based on our findings, we suggest that all chromalveolates with 4 membrane bound plastids express an ERAD-derived machinery in the PPM of their secondary plastid, coexisting physically and systematically adjacent to the host's own ERAD system.We propose herewith that this system was functionally adapted to mediate transport of nucleus-encoded PPC/plastid preproteins from the RER into the periplastid space.

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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.


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.
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.
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.

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Susceptibility of zoospores to UV radiation determines upper depth distribution limit of Arctic kelps : evidence through field experiments

2006, Wiencke, Christian, Roleda, Michael Y., Gruber, Ansgar, Clayton, Margaret N., Bischof, Kai

1. The UV susceptibility of zoospores of the brown seaweeds Saccorhiza dermato- dea, Alaria esculenta and Laminaria digitata (Laminariales) was determined in field experiments in June 2004 on Spitsbergen (78° 55' N, 11° 56' E).
2. Freshly released zoospores were exposed for one or two days at various water depths to ambient solar radiation, ambient solar radiation depleted of UVB radiation (UVBR) and ambient solar radiation depleted of both UVBR and UVAR. Subsequently, germination rates were determined after exposure to favourable light and temperature conditions in the laboratory.
3. The radiation regime was monitored at the water surface and in the water adjacent to the exposure platforms for the duration of the field exposure.
4. Under ambient solar radiation the tolerance of zoospores to UVR was highest in the shallow water species S. dermatodea, intermediate in the upper to mid sublittoral A. esculenta and lowest in the upper to mid sublittoral L. digitata. There was, however, no difference in the susceptibility of the zoospores to ambient solar radiation or to solar radiation depleted of UVBR.
5. The water body was relatively UV transparent especially in the upper water layers. The 1 % UVB depth mostly varied between 5.35 and 6.87 m. On one stormy day the 1 % UVB depth was only 3.57 m indicating resuspension of sediments.
6. We propose that as the zoospores are the developmental stages most susceptible to UVR their UVR tolerance is a major if not the most important factor for the determination of the upper depth distribution limit of these species on the shore. The results are discussed with respect to enhanced UVBR due to stratospheric ozone depletion.