A Gene in the Process of Endosymbiotic Transfer

BACKGROUND: The endosymbiotic birth of organelles is accompanied by massive transfer of endosymbiont genes to the eukaryotic host nucleus. In the centric diatom Thalassiosira pseudonana the Psb28 protein is encoded in the plastid genome while a second version is nuclear-encoded and possesses a bipartite N-terminal presequence necessary to target the protein into the diatom complex plastid. Thus it can represent a gene captured during endosymbiotic gene transfer. METHODOLOGY/PRINCIPAL FINDINGS: To specify the origin of nuclear- and plastid-encoded Psb28 in T. pseudonana we have performed extensive phylogenetic analyses of both mentioned genes. We have also experimentally tested the intracellular location of the nuclear-encoded Psb28 protein (nuPsb28) through transformation of the diatom Phaeodactylum tricornutum with the gene in question fused to EYFP. CONCLUSIONS/SIGNIFICANCE: We show here that both versions of the psb28 gene in T. pseudonana are transcribed. We also provide experimental evidence for successful targeting of the nuPsb28 fused with EYFP to the diatom complex plastid. Extensive phylogenetic analyses demonstrate that nucleotide composition of the analyzed genes deeply influences the tree topology and that appropriate methods designed to deal with a compositional bias of the sequences and the long branch attraction artefact (LBA) need to be used to overcome this obstacle. We propose that nuclear psb28 in T. pseudonana is a duplicate of a plastid localized version, and that it has been transferred from its endosymbiont

Light-Induced Energetic Decoupling as a Mechanism for Phycobilisome-Related Energy Dissipation in Red Algae: A Single Molecule Study

BACKGROUND: Photosynthetic organisms have developed multiple protective mechanisms to prevent photodamage in vivo under high-light conditions. Cyanobacteria and red algae use phycobilisomes (PBsomes) as their major light-harvesting antennae complexes. The orange carotenoid protein in some cyanobacteria has been demonstrated to play roles in the photoprotective mechanism. The PBsome-itself-related energy dissipation mechanism is still unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here, single-molecule spectroscopy is applied for the first time on the PBsomes of red alga Porphyridium cruentum, to detect the fluorescence emissions of phycoerythrins (PE) and PBsome core complex simultaneously, and the real-time detection could greatly characterize the fluorescence dynamics of individual PBsomes in response to intense light. CONCLUSIONS/SIGNIFICANCE: Our data revealed that strong green-light can induce the fluorescence decrease of PBsome, as well as the fluorescence increase of PE at the first stage of photobleaching. It strongly indicated an energetic decoupling occurring between PE and its neighbor. The fluorescence of PE was subsequently observed to be decreased, showing that PE was photobleached when energy transfer in the PBsomes was disrupted. In contrast, the energetic decoupling was not observed in either the PBsomes fixed with glutaraldehyde, or the mutant PBsomes lacking B-PE and remaining b-PE. It was concluded that the energetic decoupling of the PBsomes occurs at the specific association between B-PE and b-PE within the PBsome rod. Assuming that the same process occurs also at the much lower physiological light intensities, such a decoupling process is proposed to be a strategy corresponding to PBsomes to prevent photodamage of the photosynthetic reaction centers. Finally, a novel photoprotective role of gamma-subunit-containing PE in red algae was discussed

Genome of the red alga Porphyridium purpureum

The limited knowledge we have about red algal genomes comes from the highly specialized extremophiles, Cyanidiophyceae. Here, we describe the first genome sequence from a mesophilic, unicellular red alga, Porphyridium purpureum. The 8,355 predicted genes in P. purpureum, hundreds of which are likely to be implicated in a history of horizontal gene transfer, reside in a genome of 19.7 Mbp with 235 spliceosomal introns. Analysis of light-harvesting complex proteins reveals a nuclear-encoded phycobiliprotein in the alga. We uncover a complex set of carbohydrate-active enzymes, identify the genes required for the methylerythritol phosphate pathway of isoprenoid biosynthesis, and find evidence of sexual reproduction. Analysis of the compact, function-rich genome of P. purpureum suggests that ancestral lineages of red algae acted as mediators of horizontal gene transfer between prokaryotes and photosynthetic eukaryotes, thereby significantly enriching genomes across the tree of photosynthetic life

Algae as Protein Factories: Expression of a Human Antibody and the Respective Antigen in the Diatom Phaeodactylum tricornutum

Microalgae are thought to offer great potential as expression system for various industrial, therapeutic and diagnostic recombinant proteins as they combine high growth rates with all benefits of eukaryotic expression systems. Moreover, microalgae exhibit a phototrophic lifestyle like land plants, hence protein expression is fuelled by photosynthesis, which is CO2-neutral and involves only low production costs. So far, however, research on algal bioreactors for recombinant protein expression is very rare calling for further investigations in this highly promising field. In this study, we present data on the expression of a monoclonal human IgG antibody against the Hepatitis B surface protein and the respective antigen in the diatom Phaeodactylum tricornutum. Antibodies are fully-assembled and functional and accumulate to 8.7% of total soluble protein, which complies with 21 mg antibody per gram algal dry weight. The Hepatitis B surface protein is functional as well and is recognized by algae-produced and commercial antibodies

Mutations in Arabidopsis YCF20-like genes affect thermal dissipation of excess absorbed light energy

Plants dissipate excess absorbed light energy as heat to protect themselves from photo-oxidative stress. The Arabidopsis thaliananpq6 mutant affected in thermal dissipation was identified by its partial defect in the induction of nonphotochemical quenching of chlorophyll fluorescence (NPQ) by excess light. Positional cloning revealed that npq6 contains a frameshift mutation caused by a single base-pair deletion in the At5g43050 gene, which encodes a member of the hypothetical chloroplast open reading frame 20 (YCF20) family of proteins with unknown function(s). The YCF20 protein family is mostly conserved in oxygenic photosynthetic organisms including cyanobacteria, eukaryotic algae, and plants. Amino acid sequence comparison identified two other genes in Arabidopsis that encode similar proteins to NPQ6: At1g65420 and At3g56830. These three Arabidopsis proteins have functional chloroplast-targeting transit peptides. Using reverse genetics, a mutant with a T-DNA insertion within the At1g65420 gene was identified and shown to exhibit a low NPQ phenotype similar to that of npq6; therefore, At1g65420 was named NPQ7. In contrast, a knockdown mutant in the At3g56830 gene with lower transcript levels showed wild-type levels of NPQ. The npq6 npq7 double mutant had an additive NPQ defect, indicating that the YCF20 family members in Arabidopsis have overlapping functions affecting thermal dissipation

A Single Peroxisomal Targeting Signal Mediates Matrix Protein Import in Diatoms

Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1

Evolution of light-harvesting complex proteins from Chl c-containing algae

<p>Abstract</p> <p>Background</p> <p>Light harvesting complex (LHC) proteins function in photosynthesis by binding chlorophyll (Chl) and carotenoid molecules that absorb light and transfer the energy to the reaction center Chl of the photosystem. Most research has focused on LHCs of plants and chlorophytes that bind Chl <it>a </it>and <it>b </it>and extensive work on these proteins has uncovered a diversity of biochemical functions, expression patterns and amino acid sequences. We focus here on a less-studied family of LHCs that typically bind Chl <it>a </it>and <it>c</it>, and that are widely distributed in Chl <it>c</it>-containing and other algae. Previous phylogenetic analyses of these proteins suggested that individual algal lineages possess proteins from one or two subfamilies, and that most subfamilies are characteristic of a particular algal lineage, but genome-scale datasets had revealed that some species have multiple different forms of the gene. Such observations also suggested that there might have been an important influence of endosymbiosis in the evolution of LHCs.</p> <p>Results</p> <p>We reconstruct a phylogeny of LHCs from Chl <it>c</it>-containing algae and related lineages using data from recent sequencing projects to give ~10-fold larger taxon sampling than previous studies. The phylogeny indicates that individual taxa possess proteins from multiple LHC subfamilies and that several LHC subfamilies are found in distantly related algal lineages. This phylogenetic pattern implies functional differentiation of the gene families, a hypothesis that is consistent with data on gene expression, carotenoid binding and physical associations with other LHCs. In all probability LHCs have undergone a complex history of evolution of function, gene transfer, and lineage-specific diversification.</p> <p>Conclusion</p> <p>The analysis provides a strikingly different picture of LHC diversity than previous analyses of LHC evolution. Individual algal lineages possess proteins from multiple LHC subfamilies. Evolutionary relationships showed support for the hypothesized origin of Chl <it>c </it>plastids. This work also allows recent experimental findings about molecular function to be understood in a broader phylogenetic context.</p

Silencing of the Violaxanthin De-Epoxidase Gene in the Diatom Phaeodactylum tricornutum Reduces Diatoxanthin Synthesis and Non-Photochemical Quenching

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

Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropis gaditana

The potential use of algae in biofuels applications is receiving significant attention. However, none of the current algal model species are competitive production strains. Here we present a draft genome sequence and a genetic transformation method for the marine microalga Nannochloropsis gaditana CCMP526. We show that N. gaditana has highly favourable lipid yields, and is a promising production organism. The genome assembly includes nuclear (~29 Mb) and organellar genomes, and contains 9,052 gene models. We define the genes required for glycerolipid biogenesis and detail the differential regulation of genes during nitrogen-limited lipid biosynthesis. Phylogenomic analysis identifies genetic attributes of this organism, including unique stramenopile photosynthesis genes and gene expansions that may explain the distinguishing photoautotrophic phenotypes observed. The availability of a genome sequence and transformation methods will facilitate investigations into N. gaditana lipid biosynthesis and permit genetic engineering strategies to further improve this naturally productive alga