Controlled supply of CO2 to batch cultures of the diatom Phaeodactylum tricornutum
2017, Gruber, Ansgar, Kroth, Peter G., Yu, Guilan
For the growth of photosynthetic organisms, supply of CO2 is essential. Experimental work on the uptake and utilisation of inorganic carbon, requires that CO2 concentrations can be adjusted and kept stable. Here we tested the suitability of a culture method that allows supply of CO2 to a cell suspension, without the need of a continuous external gas supply for experimental work with the diatom Phaeodactylum tricornutum. This approach utilizes buffers with different ratios of HCO3-/CO32- in one chamber of a two-tier vessel, releasing different amounts of CO2 to the gas phase of the vessel, which is shared with the cell culture in the other chamber of the vessel. We cultured P. tricornutum under three different CO2 concentrations, while monitoring cell density, CO2 concentration in the gas phase, and pH within the cultures. We found the method very useful for work with P. tricornutum and found that the method also allows the creation of CO2 deplete conditions. This culturing system, while not as precise as a chemostat culture with supply of gas mixtures containing CO2, is simple to use and offers the possibility to adjust CO2 growth conditions.
Protein targeting into complex plastids : support for the trans-locator model
2011, Vugrinec, Sascha, Gruber, Ansgar, Kroth, Peter G.
Plastids of diatoms are surrounded by four membranes. The outermost membrane is continuous with the endoplasmic reticulum and therefore is termed chloroplast ER (CER) membrane. The complex ultra structure of diatom plastids naturally requires more transport steps to import nucleus encoded proteins into the plastid compared to higher plant plastids which possess only two envelope membranes.
Several hypothetic models for the import of preproteins into the complex plastids of diatoms are discussed. Common to all these models is the postulation of a first cotranslational transport step into the chloroplast endoplasmic reticulum lumen via the Sec61 translocon. Furthermore, all models postulate transport via a translocator in the innermost membrane similar to the Tic complex (translocon of the inner chloroplasts envelope) of higher plant plastids. The models differ, however, with respect to their explanation of transport out of the CERlumen and into the interenvelope space: either translocators, vesicles crossing the periplastidic space or putative membrane channels connecting CERlumen and the interenvelope space have been proposed. To investigate the presence of such a hypothetic connection between the CERlumen and the interenvelope space, we expressed different preproteins in the diatom Phaeodactylum tricornutum that were fused to selfassembling fragments of GFP (GFP110 and GFP11). Complementary fragments were fused to marker proteins of the CERlumen and the interenvelope space, respectively. Our data indicate that the GFP110 and GFP11 fusion proteins are located in two separate compartments which are not connected to each other.
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.
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.
Analysing size variation during light-starvation response of nutritionally diverse chrysophytes with a Coulter counter
2013, Rottberger, Julia, Gruber, Ansgar, Kroth, Peter G.
Mixotrophy is a combination of autotrophic and heterotrophic nutrition. The regulation of the nutritional mode in mixotrophic algae is not yet fully understood. Changes in the light regime are likely to affect cell size of autotrophic, mixotrophic and heterotrophic chrysophytes to a varying extend. Here we demonstrate that an automatic particle analyser (Coulter counter) is a helpful tool not only for cell counts, but also for the detection of changes in mean cell size, total cell bio-volume or size distribution of chrysophyte cultures. The method is time saving and therefore allows large comparative studies, however, the method tends to underestimate the absolute size compared to microscopic measurements. The limitations of automated particle analysis opposed to traditional microscopical measurements are discussed exemplarily for Dinobryon divergens.
Sporogenesis under ultraviolet radiation in laminaria digitata (phaeophyceae) reveals protection of photosensitive meiospores within soral tissue : physiological and anatomical evidence
2011, Gruber, Ansgar, Roleda, Michael Y., Bartsch, Inka, Hanelt, Dieter, Wiencke, Christian
To study the effect of different radiation conditions on sporogenesis of Laminaria digitata (Huds.) J. V. Lamour., excised disks were induced to form sporangia under PAR (P), PAR + ultraviolet-A (UVA) (PA), and PAR + UVA + ultraviolet-B (UVB) (PAB) conditions in the laboratory. Vitality of meiospores, released from sori induced under different radiation conditions in the laboratory and from sori of wild sporophytes acclimated to in situ solar radiation in the presence and absence of ultraviolet radiation (UVR), was measured in terms of their germination capacity. Sorus induction in disks of laboratory-grown sporophytes was not hampered under light supplemented with UVR, and sorus area was not significantly different among P, PA, and PAB. Vitality and germination rate of meiospores released from sori induced under different radiation treatments was comparable. Likewise, screening of UVR of the natural solar radiation did not promote higher germination rates of meiospores released from wild sporophytes. Germination rates were, however, higher in meiospores released from laboratory-induced sori compared to sori of wild sporophytes. Higher DNA damage (formation of cyclobutane pyrimidine dimers, CPDs) was observed in laboratory-grown nonsorus compared to sorus tissue, while CPDs were nondetectable in both sorus and nonsorus tissue of wild sporophytes. To explain the apparent protection of developing meiospores and the unexpected UV resistance of soral tissue, concurrent anatomical investigations of sporogenic tissue were performed. We observed the previously unreported existence of two types of sterile paraphysis cells. One type of paraphysis cells, the most frequent type, contained several red-fluorescing plastids. The other type, less frequently occurring, was completely filled with substances emitting blue fluorescence under violet excitation, presumably brown algal phenolic compounds (phlorotannins). Cells of this type were irregularly scattered within the sorus and did not contain red-fluorescing plastids. Meiospore-containing sporangia were positioned embedded between both types of paraphysis cells. In vegetative tissue, blue autofluorescence was observed only in injured parts of the blade. Results of our study suggest that the sorus structure with phlorotannins localized in the specialized paraphysis cells may be able to screen harmful UVR and protect UV-sensitive meiospores inside the sporangia.
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.
Influence of nutrients and light on autotrophic, mixotrophic and heterotrophic freshwater chrysophytes
2013, Rottberger, Julia, Gruber, Ansgar, Boenigk, Jens, Kroth, Peter G.
Chrysophyte algae show wide variation in their nutritional modes, which can be especially advantageous in oligotrophic conditions. However, the capacities and strategies of the algae to adapt to changing conditions may vary due to different nutritional requirements of individual species. In this context, comparative analyses evaluating the physiological range of mixotrophic chrysophytes are important to predict possible changes in phytoplankton composition. We compared 4 freshwater chrysophytes—Poteriospumella lacustris, Poterioochromonas malhamensis, Dinobryon divergens and Mallomonas annulata—under different growth conditions. The diatom Phaeodactylum tricornutum served as a photoautotrophic reference strain. We demonstrate active growth of P. lacustris and the mixotrophic P. malhamensis under chemoheterotrophic conditions. Neither of the chrysophytes were growing photoautotrophically; however, P. malhamensis showed some photosynthetic activity and survived longer when kept in the light. M. annulata, P. tricornutum and the mixotrophic D. divergens showed light-limited growth when kept in inorganic media. Even though D. divergens consumed living bacteria, it did not grow in the dark in the presence of the bacteria only. We detected large differences in the general nutritional preferences between the 2 mixotrophs in relation to the nutritional features of all investigated strains.
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.