A partial mitochondrial glycolysis in Phaeodactylum tricornutum and other Heterokonts: Verification of compartmentalisation using the Green Fluorescent Protein and molecular phylogeny

Die Glykolyse ist einer der ältesten Stoffwechselwege, und die daran beteiligten Enzyme sind universell in Eukaryoten, Eubakterien und Archaeabakterien vorhanden. Während die Glykolyse in Eukaryoten normalerweise im Cytosol lokalisiert ist, konnte bereits in einer früheren Arbeit die Präsenz eines bifunktionellen Fusionsproteins aus den zwei glykolytischen Enzymen Triosephosphatisomerase (TPI) und Glycerinaldehyd-3-Phosphatdehydrogenase (GAPDH), welches in die Mitochondrien der Diatomee Phaeodactylum tricornutum (ein Heterokont) importiert wird, nachgewiesen werden (Liaud et al. 2000). Auch Oomyceten und Braunalgen (beides Heterokonten) besitzen diese bifunktionelle TPI-GAPDH. Im Rahmen der vorliegenden Arbeit wurde ein bereits existierendes Transformationssystems für Phaeodactylum (Apt et al. 1996, Zaslavskaia et al. 2000) in der Arbeitsgruppe etabliert und mittels GFP gekoppelter in vivo Expression die intrazelluläre Lokalisierung von Präsequenzen glykolytischer Proteine bestimmt. Für je eine Isoform der Triosephosphatisomerase, Glycerinaldehyd-3-Phosphatdehydrogenase, Phosphoglyceratkinase (PGK), Phosphoglyceratmutase (PGM), Enolase (ENO) und Pyruvatkinase (PYK) aus Phaeodactylum konnte hierbei ein Import in die Mitochondrien gezeigt werden. In heterologen Ansätzen wurden zudem mitochondriale Isoformen des TPI-GAPDH Fusionsproteins, der Phosphoglyceratmutase und der Pyruvatkinase aus Oomyceten bestimmt, während z.B. in den Braunalgen bisher nur eine mitochondriale TPI-GAPDH identifiziert werden konnte. Die phylogenetischen Analysen der GAPDH, der PGK und der PYK zeigen, dass die mitochondrialen Isoformen der Heterokonten einen gemeinsamen Ursprung haben und sie schon vor der Akquirierung des Plastids in einem gemeinsamen Vorfahren vorhanden waren. Diese partielle Glykolyse, die zusätzlich zu der Glykolyse im Cytosol existiert, kommt wahrscheinlich bei allen Heterokonten vor und könnte ein Merkmal dieser Gruppe sein.Glycolysis is one of the most ancient metabolic pathways; the enzymes of this pathway are ubiquitous and are present in Eukaryotes, Eubacteria and Archaebacteria alike. While glycolysis in Eukaryotes usually takes place in the cytosol, the presence of a bi-functional fusion protein consisting of the enzymes Triosephosphate Isomerase (TPI) and Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) has been shown in mitochondria of the Diatom Phaeodactylum tricornutum (a Heterokont) in a previous study (Liaud et al. 2000). Oomycetes and brown algae (both Heterokonts) also possess this bi-functional TPI-GAPDH. In this work, an existing transformation system for Phaeodactylum (Apt et al. 1996, Zaslavskaia et al. 2000) was established in our laboratory and the intracellular localisation of several pre-sequences of putative mitochondrial glycolytic proteins was investigated using GFP tagged in vivo expression. Import into the mitochondrium was demonstrated for isoforms of Triosephosphate Isomerase, Glyceraldehyde-3-phosphatedehydrogenase, Phosphoglycerate Kinase (PGK), Phosphoglycerate Mutase (PGM), Enolase (ENO), and Pyruvate Kinase (PYK) from Phaeodactylum. Furthermore, using a heterologous approach, mitochondrial isoforms of the TPI-GAPDH fusion protein, Phosphoglycerate Mutase and Pyruvate Kinase from Oomycetes were determined, while for brown algae only the mitochondrial TPI-GAPDH could be identified so far. Phylogenetic analysis of the Glyceraldehyde-3-phosphatedehydrogenase, Phosphoglycerate Kinase, and Pyruvate Kinase shows that the mitochondrial isoforms from Heterokonts have a common origin, and that the genes for these enzymes existed in a common ancestor before the acquisition of the plastid. This partial glycolysis, which exists in addition to the cytosolic glycolysis, probably occurs in all Heterokonts and could be characteristic for this group

Blasticidin-S deaminase, a new selection marker for genetic transformation of the diatom Phaeodactylum tricornutum

Most genetic transformation protocols for the model diatom Phaeodactylum tricornutum rely on one of two available antibiotics as selection markers: Zeocin (a formulation of phleomycin D1) or nourseothricin. This limits the number of possible consecutive genetic transformations that can be performed. In order to expand the biotechnological possibilities for P. tricornutum, we searched for additional antibiotics and corresponding resistance genes that might be suitable for use with this diatom. Among the three different antibiotics tested in this study, blasticidin-S and tunicamycin turned out to be lethal to wild-type cells at low concentrations, while voriconazole had no detectable effect on P. tricornutum. Testing the respective resistance genes, we found that the blasticidin-S deaminase gene (bsr) effectively conferred resistance against blasticidin-S to P. tricornutum. Furthermore, we could show that expression of bsr did not lead to cross-resistances against Zeocin or nourseothricin, and that genetically transformed cell lines with resistance against Zeocin or nourseothricin were not resistant against blasticidin-S. In a proof of concept, we also successfully generated double resistant (against blasticidin-S and nourseothricin) P. tricornutum cell lines by co-delivering the bsr vector with a vector conferring nourseothricin resistance to wild-type cells

Plastid thylakoid architecture optimizes photosynthesis in diatoms

Photosynthesis is a unique process that allows independent colonization of the land by plants and of the oceans by phytoplankton. Although the photosynthesis process is well understood in plants, we are still unlocking the mechanisms evolved by phytoplankton to achieve extremely efficient photosynthesis. Here, we combine biochemical, structural and in vivo physiological studies to unravel the structure of the plastid in diatoms, prominent marine eukaryotes. Biochemical and immunolocalization analyses reveal segregation of photosynthetic complexes in the loosely stacked thylakoid membranes typical of diatoms. Separation of photosystems within subdomains minimizes their physical contacts, as required for improved light utilization. Chloroplast 3D reconstruction and in vivo spectroscopy show that these subdomains are interconnected, ensuring fast equilibration of electron carriers for efficient optimum photosynthesis. Thus, diatoms and plants have converged towards a similar functional distribution of the photosystems although via different thylakoid architectures, which likely evolved independently in the land and the ocean.ISSN:2041-172

Diatom Vacuolar 1,6-β-Transglycosylases can Functionally Complement the Respective Yeast Mutants

Diatoms are unicellular photoautotrophic algae, which can be found in any aquatic habitat. The main storage carbohydrate of diatoms is chrysolaminarin, a nonlinear β-glucan, consisting of a linear 1,3-β-chain with 1,6-β-branches, which is stored in cytoplasmic vacuoles. The metabolic pathways of chrysolaminarin synthesis in diatoms are poorly investigated, therefore we studied two potential 1,6-β-transglycosylases (TGS) of the diatom Phaeodactylum tricornutum which are similar to yeast Kre6 proteins and which potentially are involved in the branching of 1,3-β-glucan chains by adding d-glucose as 1,6-side chains. We genetically fused the full-length diatom TGS proteins to GFP and expressed these constructs in P. tricornutum, demonstrating that the enzymes are apparently located in the vacuoles, which indicates that branching of chrysolaminarin may occur in these organelles. Furthermore, we demonstrated the functionality of the diatom enzymes by expressing TGS1 and 2 proteins in yeast, which resulted in a partial complementation of growth deficiencies of a transglycosylase-deficient ∆kre6 yeast strain.publishe

Comprehensive computational analysis of leucine-rich repeat (LRR) proteins encoded in the genome of the diatom Phaeodactylum tricornutum

We have screened the genome of the marine diatom Phaeodactylum tricornutum for gene models encoding proteins exhibiting leucine-rich repeat (LRR) structures. In order to reveal the functionality of these proteins, their amino acid sequences were scanned for known domains and for homologies to other proteins. Additionally, proteins were categorized into different LRR-families according to the variable sequence part of their LRR. This approach enabled us to group proteins with potentially similar functionality and to classify also LRR proteins where no characterized homologues in other organisms exist. Most interestingly, we were able to indentify several transmembrane LRR-proteins, which are likely to function as receptor-like molecules. However, none of them carry additional domains that are typical for mammalian or plant-like receptors. Thus, the respective signal recognition pathways seem to be substantially different in diatoms. Moreover, P. tricornutum encodes a family of secreted LRR proteins likely to function as adhesion or binding proteins as part of the extracellular matrix. Additionally, intracellular LRR-only proteins were divided into proteins similar to RasGTPase activators, regulators of nuclear transport, and mitotic regulation. Our approach allowed us to draw a detailed picture of the conservation and diversification of LRR proteins in the marine diatom P. tricornutum

Rapid induction of GFP expression by the nitrate reductase promoter in the diatom Phaeodactylum tricornutum

ABSTRACT An essential prerequisite for a controlled transgene expression is the choice of a suitable promoter. In the model diatom Phaeodactylum tricornutum, the most commonly used promoters for trans-gene expression are the light dependent lhcf1 promoters (derived from two endogenous genes encoding fucoxanthin chlorophyll a/c binding proteins) and the nitrate dependent nr promoter (derived from the endogenous nitrate reductase gene). In this study, we investigated the time dependent expression of the green fluorescent protein (GFP) reporter under control of the nitrate reductase promoter in independently genetically transformed P. tricornutum cell lines following induction of expression by change of the nitrogen source in the medium via flow cytometry, microscopy and western blotting. In all investigated cell lines, GFP fluorescence started to increase 1 h after change of the medium, the fastest increase rates were observed between 2 and 3 h. Fluorescence continued to increase slightly for up to 7 h even after transfer of the cells to ammonium medium. The subsequent decrease of GFP fluorescence was much slower than the increase, probably due to the stability of GFP. The investigation of several cell lines transformed with nr based constructs revealed that, also in the absence of nitrate, the promoter may show residual activity. Furthermore, we observed a strong variation of gene expression between independent cell lines, emphasising the importance of a thorough characterisation of genetically modified cell lines and their individual expression patterns

Rapid induction of GFP expression by the nitrate reductase promoter in the diatom Phaeodactylum tricornutum

An essential prerequisite for a controlled transgene expression is the choice of a suitable promoter. In the model diatom Phaeodactylum tricornutum, the most commonly used promoters for trans-gene expression are the light dependent lhcf1 promoters (derived from two endogenous genes encoding fucoxanthin chlorophyll a/c binding proteins) and the nitrate dependent nr promoter (derived from the endogenous nitrate reductase gene). In this study, we investigated the time dependent expression of the green fluorescent protein (GFP) reporter under control of the nitrate reductase promoter in independently genetically transformed P. tricornutum cell lines following induction of expression by change of the nitrogen source in the medium via flow cytometry, microscopy and western blotting. In all investigated cell lines, GFP fluorescence started to increase 1 h after change of the medium, the fastest increase rates were observed between 2 and 3 h. Fluorescence continued to increase slightly for up to 7 h even after transfer of the cells to ammonium medium. The subsequent decrease of GFP fluorescence was much slower than the increase, probably due to the stability of GFP. The investigation of several cell lines transformed with nr based constructs revealed that, also in the absence of nitrate, the promoter may show residual activity. Furthermore, we observed a strong variation of gene expression between independent cell lines, emphasising the importance of a thorough characterisation of genetically modified cell lines and their individual expression patterns

Organelle Studies and Proteome Analyses on Mitochondria and Plastids Fractions from the Diatom Thalassiosira pseudonana

Diatoms are unicellular algae and evolved by secondary endosymbiosis, a process in which a red alga-like eukaryote was engulfed by a heterotrophic eukaryotic cell. This gave rise to plastids of remarkable complex architecture and ultrastructure that require elaborate protein importing, trafficking, signaling and intracellular cross-talk pathways. Studying both plastids and mitochondria and their distinctive physiological pathways in organello may greatly contribute to our understanding of photosynthesis, mitochondrial respiration, and diatom evolution. The isolation of such complex organelles, however, is still demanding, and existing protocols are either limited to a few species (for plastids) or have not been reported for diatoms so far (for mitochondria). In this work, we present the first isolation protocol for mitochondria from the model diatom Thalassiosira pseudonana. Apart from that, we extended the protocol so that it is also applicable for the purification of a high-quality plastids fraction, and provide detailed structural and physiological characterizations of the resulting organelles. Isolated mitochondria were structurally intact, showed clear evidence of mitochondrial respiration, but the fractions still contained residual cell fragments. In contrast, plastid isolates were virtually free of cellular contaminants, featured structurally preserved thylakoids performing electron transport, but lost most of their stromal components as concluded from western blots and mass spectrometry. LC-ESI-MS/MS studies on mitochondria and thylakoids, moreover, allowed detailed proteome analyses which resulted in extensive proteome maps for both plastids and mitochondria thus helping us to broaden our understanding of organelle metabolism and functionality in diatoms.publishe

Reduced vacuolar β-1,3-glucan synthesis affects carbohydrate metabolism as well as plastid homeostasis and structure in Phaeodactylum tricornutum

The β-1,3-glucan chrysolaminarin is the main storage polysaccharide of diatoms. In contrast to plants and green algae, diatoms and most other algal groups do not accumulate storage polysaccharides in their plastids. The diatom Phaeodactylum tricornutum possesses only a single gene encoding a putative β-1,3-glucan synthase (PtBGS). Here, we characterize this enzyme by expressing GFP fusion proteins in P. tricornutum and by creating and investigating corresponding gene silencing mutants. We demonstrate that PtBGS is a vacuolar protein located in the tonoplast. Metabolite analyses of two mutant strains with reduced amounts of PtBGS reveal a reduction in their chrysolaminarin content and an increase of soluble sugars and lipids. This indicates that carbohydrates are shunted into alternative pathways when chrysolaminarin production is impaired. The mutant strains show reduced growth and lower photosynthetic capacities, while possessing higher photoprotective abilities than WT cells. Interestingly, a strong reduction in PtBGS expression also results in aberrations of the usually very regular thylakoid membrane patterns, including increased thylakoid thickness, reduced numbers of thylakoids per plastid, and increased numbers of lamellae per thylakoid stack. Our data demonstrate the complex intertwinement of carbohydrate storage in the vacuoles with carbohydrate metabolism, photosynthetic homeostasis, and plastid morphology.publishe

The intracellular distribution of inorganic carbon fixing enzymes does not support the presence of a C4 pathway in the diatom Phaeodactylum tricornutum

Diatoms are unicellular algae and important primary producers. The process of carbon fixation in diatoms is very efficient even though the availability of dissolved CO2 in sea water is very low. The operation of a carbon concentrating mechanism (CCM) also makes the more abundant bicarbonate accessible for photosynthetic carbon fixation. Diatoms possess carbonic anhydrases as well as metabolic enzymes potentially involved in C4 pathways; however, the question as to whether a C4 pathway plays a general role in diatoms is not yet solved. While genome analyses indicate that the diatom Phaeodactylum tricornutum possesses all the enzymes required to operate a C4 pathway, silencing of the pyruvate orthophosphate dikinase (PPDK) in a genetically transformed cell line does not lead to reduced photosynthetic carbon fixation. In this study, we have determined the intracellular location of all enzymes potentially involved in C4-like carbon fixing pathways in P. tricornutum by expression of the respective proteins fused to green fluorescent protein (GFP), followed by fluorescence microscopy. Furthermore, we compared the results to known pathways and locations of enzymes in higher plants performing C3 or C4 photosynthesis. This approach revealed that the intracellular distribution of the investigated enzymes is quite different from the one observed in higher plants. In particular, the apparent lack of a plastidic decarboxylase in P. tricornutum indicates that this diatom does not perform a C4-like CCM.publishe