Molecular Characterisation of Diatom Plastids

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.