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 (ASA-FAP), 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.

Diatoms contain a highly flexible capacity to dissipate excessively absorbed light by nonphotochemical fluorescence quenching (NPQ) based on the light-induced conversion of diadinoxanthin (Dd) into diatoxanthin (Dt) and the presence of Lhcx proteins. Their NPQ fine regulation on the molecular level upon a shift to dynamic light conditions is unknown. We investigated the regulation of Dd + Dt amount, Lhcx gene and protein synthesis and NPQ capacity in the diatom Phaeodactylum tricornutum after a change from continuous low light to 3 d of sine (SL) or fluctuating (FL) light conditions. Four P. tricornutum strains with different NPQ capacities due to different expression of Lhcx1 were included. All strains responded to dynamic light comparably, independently of initial NPQ capacity. During SL, NPQ capacity was strongly enhanced due to a gradual increase of Lhcx2 and Dd + Dt amount. During FL, cells enhanced their NPQ capacity on the first day due to increased Dd + Dt, Lhcx2 and Lhcx3; already by the second day light acclimation was accomplished. While quenching efficiency of Dt was strongly lowered during SL conditions, it remained high throughout the whole FL exposure. Our results highlight a more balanced and cost-effective photoacclimation strategy of P. tricornutum under FL than under SL conditions.

Diatoms are a major group of primary producers ubiquitous in all aquatic ecosystems. To protect themselves from photooxidative damage in a fluctuating light climate potentially punctuated with regular excess light exposures, diatoms have developed several photoprotective mechanisms. The xanthophyll cycle (XC) dependent non-photochemical chlorophyll fluorescence quenching (NPQ) is one of the most important photoprotective processes that rapidly regulate photosynthesis in diatoms. NPQ depends on the conversion of diadinoxanthin (DD) into diatoxanthin (DT) by the violaxanthin de-epoxidase (VDE), also called DD de-epoxidase (DDE). To study the role of DDE in controlling NPQ, we generated transformants of P. tricornutum in which the gene (Vde/Dde) encoding for DDE was silenced. RNA interference was induced by genetic transformation of the cells with plasmids containing either short (198 bp) or long (523 bp) antisense (AS) fragments or, alternatively, with a plasmid mediating the expression of a self-complementary hairpin-like construct (inverted repeat, IR). The silencing approaches generated diatom transformants with a phenotype clearly distinguishable from wildtype (WT) cells, i.e. a lower degree as well as slower kinetics of both DD de-epoxidation and NPQ induction. Real-time PCR based quantification of Dde transcripts revealed differences in transcript levels between AS transformants and WT cells but also between AS and IR transformants, suggesting the possible presence of two different gene silencing mediating mechanisms. This was confirmed by the differential effect of the light intensity on the respective silencing efficiency of both types of transformants. The characterization of the transformants strengthened some of the specific features of the XC and NPQ and confirmed the most recent mechanistic model of the DT/NPQ relationship in diatoms.

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.

BackgroundLight, the driving force of photosynthesis, can be harmful when present in excess; therefore, any light harvesting system requires photoprotection. Members of the extended light-harvesting complex (LHC) protein superfamily are involved in light harvesting as well as in photoprotection and are found in the red and green plant lineages, with a complex distribution pattern of subfamilies in the different algal lineages.ResultsHere, we demonstrate that the recently discovered “red lineage chlorophyll a/b-binding-like proteins” (RedCAPs) form a monophyletic family within this protein superfamily. The occurrence of RedCAPs was found to be restricted to the red algal lineage, including red algae (with primary plastids) as well as cryptophytes, haptophytes and heterokontophytes (with secondary plastids of red algal origin). Expression of a full-length RedCAP:GFP fusion construct in the diatom Phaeodactylum tricornutum confirmed the predicted plastid localisation of RedCAPs. Furthermore, we observed that similarly to the fucoxanthin chlorophyll a/c-binding light-harvesting antenna proteins also RedCAP transcripts in diatoms were regulated in a diurnal way at standard light conditions and strongly repressed at high light intensities.ConclusionsThe absence of RedCAPs from the green lineage implies that RedCAPs evolved in the red lineage after separation from the the green lineage. During the evolution of secondary plastids, RedCAP genes therefore must have been transferred from the nucleus of the endocytobiotic alga to the nucleus of the host cell, a process that involved complementation with pre-sequences allowing import of the gene product into the secondary plastid bound by four membranes. Based on light-dependent transcription and on localisation data, we propose that RedCAPs might participate in the light (intensity and quality)-dependent structural or functional reorganisation of the light-harvesting antennae of the photosystems upon dark to light shifts as regularly experienced by diatoms in nature. Remarkably, in plastids of the red lineage as well as in green lineage plastids, the phycobilisome based cyanobacterial light harvesting system has been replaced by light harvesting systems that are based on members of the extended LHC protein superfamily, either for one of the photosystems (PS I of red algae) or for both (diatoms). In their proposed function, the RedCAP protein family may thus have played a role in the evolutionary structural remodelling of light-harvesting antennae in the red lineage.