Molecular Characterisation of Diatom Plastids

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2008
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Molekulare Charakterisierung von Kieselalgen-Plastiden
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Abstract
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.
Summary in another language
Kieselalgen (Diatomeen) sind photoautotrophe, einzellige Organismen die in vielen aquatischen Habitaten zu finden sind. Kieselalgen-Plastiden sind vermutlich durch sekundäre Endocytobiose entstanden, also durch die Aufnahme einer eukaryotischen Alge in eine eukaryotische Zelle mit anschließender evolutionärer Reduzierung und Spezialisierung des Endosymbionten zum Zellorganell. Kieselalgen-Plastiden weisen daher einige Besonderheiten gegenüber Plastiden höherer Pflanzen auf, beispielsweise sind sie von vier (statt zwei) Hüllmembranen umgeben.
Plastiden und Mitochondrien enthalten eigene Genome, die auf die Genome ihrer freilebenden Vorfahren, Cyanobakterien bzw. Alphaproteobakterien, zurückgehen. Das Verhältnis der von Plastiden- und Mitochiondriengenom-Kopien zu Kerngenom-Kopien in der Diatomee Phaeodactylum tricornutum konnte mittels quantitativer Echtzeit-Polymerase-Kettenreaktion (quantitative real-time PCR, qPCR) bestimmt werden. Fusion einer plastidären Rekombinase (RecA) mit dem grünen fluoreszierenden Protein (green fluorescent protein, GFP) führt zur selektiven Markierung plastidärer Nucleoide der Diatomee Phaeodactylum tricornutum.
Viele essentielle Enzyme der Photosynthese sind im Kerngenom codiert, die Genprodukte müssen daher in die Plastiden importiert werden. Besonders wichtig ist dabei ein konserviertes Sequenz-Motiv unbekannter Funktion ( ASAFAP -Motiv) innerhalb der N-terminalen Präsequenz von Plastiden-Präproteinen. Durch die molekulare Charakterisierung des konservierten Präsequenz-Motivs sind Rückschlüsse auf den Import Mechanismus und eine bessere Vorhersage plastidär lokalisierter Proteinen im genomischen Maßstab möglich.
Die Genome der Kieselalgen Modellorganismen P. tricornutum und Thalassiosira pseudonana sind vollständig sequenziert. Durch Analyse der wahrscheinlichen intrazellulären Lokalisationen von Enzymen aufgrund ihrer identifizierten Präsequenzen konnten Hinweise auf eine C4-artige Photosynthese in P. tricornutum gefunden werden, sowie Modelle für Kohlenstoff-Konzentrierungs-Mechanismen und CO2-Fixierung in P. tricornutum und T. pseudonana entworfen werden. Auffällig ist auch das Auftreten mehrfacher Isoformen von Enzymen des Calvin Zyklus und der Glykolyse in den untersuchten Kieselalgen. Die Isoformen unterscheiden sich in ihren Präsequenzen und sind vermutlich in Plastiden, Mitochondrien und dem Cytosol aktiv.
Der Austausch von Stoffwechselprodukten zwischen Stroma, Cytosol und anderen Organellen ist entscheidend für die Funktion von Plastiden. Acht bzw. sechs putative plastidäre Nukleotid Transporter sind in den Genomen von T. pseudonana und P. tricornutum codiert. Fusionsproteine der Präsequenzen oder ganzer Nukleotidtransporter mit GFP zeigen, dass die untersuchten Nukleotidtransporter Plastiden-assoziiert sind.
Subject (DDC)
570 Biosciences, Biology
Keywords
Chloroplast,Diatom,Evolution,Genome analysis,Protein transport
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Cite This
ISO 690GRUBER, Ansgar, 2008. Molecular Characterisation of Diatom Plastids [Dissertation]. Konstanz: University of Konstanz
BibTex
@phdthesis{Gruber2008Molec-7419,
  year={2008},
  title={Molecular Characterisation of Diatom Plastids},
  author={Gruber, Ansgar},
  address={Konstanz},
  school={Universität Konstanz}
}
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