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Non-cyanobacteria diazotrophs (NCDs) were shown to dominate in surface waters shifting the long-held paradigm of cyanobacteria dominance and raising fundamental questions on how these putative heterotrophic bacteria thrive in sunlit oceans. Here, we report an unprecedented finding in the widely used model diatom Phaeodactylum tricornutum (Pt) of NCDs sustaining diatom cells in the absence of bioavailable nitrogen. We identified PtNCDs using metagenomics sequencing and detected nitrogenase gene in silico and/or by PCR. We demonstrated nitrogen fixation in PtNCDs and their close genetic affiliation with NCDs from the environment. We showed the wide occurrence of this type of symbiosis with the isolation of NCDs from other microalgae, their identification in the environment, and predicted their associations with photosynthetic microalgae. Overall, this study provides evidence for a previously overlooked symbiosis using a multidisciplinary model-based approach, which will help understand the different players driving global marine nitrogen fixation.

In many eukaryotes, such as dioicous mosses and many algae, sex is determined by UV sex chromosomes and is expressed during the haploid phase of the life cycle. In these species, the male and female developmental programs are initiated by the presence of the U- or V-specific regions of the sex chromosomes but, as in XY and ZW systems, sexual differentiation is largely driven by autosomal sex-biased gene expression. The mechanisms underlying the regulation of sex-biased expression of genes during sexual differentiation remain elusive. Here, we investigated the extent and nature of epigenomic changes associated with UV sexual differentiation in the brown alga Ectocarpus, a model UV system. Six histone modifications were quantified in near-isogenic lines, leading to the identification of 16 chromatin signatures across the genome. Chromatin signatures correlated with levels of gene expression and histone PTMs changes in males versus females occurred preferentially at genes involved in sex-specific pathways. Despite the absence of chromosome scale dosage compensation and the fact that UV sex chromosomes recombine across most of their length, the chromatin landscape of these chromosomes was remarkably different to that of autosomes. Hotspots of evolutionary young genes in the pseudoautosomal regions appear to drive the exceptional chromatin features of UV sex chromosomes.

Sedimentary ancient DNA (sedaDNA) analyses are increasingly used to reconstruct marine ecosystems. The majority of marine sedaDNA studies use a metabarcoding approach (extraction and analysis of specific DNA fragments of a defined length), targeting short taxonomic marker genes. Promising examples are 18S-V9 rRNA (~121–130 base pairs, bp) and diat-rbcL (76 bp), targeting eukaryotes and diatoms, respectively. However, it remains unknown how 18S-V9 and diat-rbcL derived compositional profiles compare to metagenomic shotgun data, the preferred method for ancient DNA analyses as amplification biases are minimised. We extracted DNA from five Santa Barbara Basin sediment samples (up to ~11 000 years old) and applied both a metabarcoding (18S-V9 rRNA, diat-rbcL) and a metagenomic shotgun approach to (i) compare eukaryote, especially diatom, composition, and (ii) assess sequence length and database related biases. Eukaryote composition differed considerably between shotgun and metabarcoding data, which was related to differences in read lengths (~112 and ~161 bp, respectively), and overamplification of short reads in metabarcoding data. Diatom composition was influenced by reference bias that was exacerbated in metabarcoding data and characterised by increased representation of Chaetoceros, Thalassiosira and Pseudo-nitzschia. Our results are relevant to sedaDNA studies aiming to accurately characterise paleo-ecosystems from either metabarcoding or metagenomic data.

Cytosine DNA methylation is an important epigenetic mark in eukaryotes that is involved in the transcriptional control of mainly transposable elements in mammals, plants, and fungi. Eukaryotes encode a diverse set of DNA methyltransferases that were iteratively acquired and lost during evolution. The Stramenopiles-Alveolate-Rhizaria (SAR) lineages are a major group of ecologically important marine microeukaryotes that include the main phytoplankton classes such as diatoms and dinoflagellates. However, little is known about the diversity of DNA methyltransferases and their role in the deposition and maintenance of DNA methylation in microalgae. We performed a phylogenetic analysis of DNA methyltransferase families found in marine microeukaryotes and show that they encode divergent DNMT3, DNMT4, DNMT5 and DNMT6 enzymes family revisiting previously established phylogenies. Furthermore, we reveal a novel group of DNMTs with three classes of enzymes within the DNMT5 family. Using a CRISPR/Cas9 strategy we demonstrate that the loss of the DNMT5 gene correlates with a global depletion of DNA methylation and overexpression of transposable elements in the model diatom Phaeodactylum tricornutum . The study provides a pioneering view of the structure and function of a DNMT family in the SAR supergroup.

Diatom dominance in contemporary aquatic environments indicates that they have developed unique and effective mechanisms to cope with the rapid and considerable fluctuations that characterize these environments. In view of their evolutionary history from a secondary endosymbiosis, inter-organellar regulation of biochemical activities may be of particular relevance. Diatom mitochondrial alternative oxidase (AOX) is believed to play a significant role in supplying chloroplasts with ATP produced in the mitochondria. Using the model diatom Phaeodactylum tricornutum we generated AOX knockdown lines, and followed sensitivity to stressors, photosynthesis and transcriptome and metabolome profiles of wild-type and knockdown lines. We show here that expression of the AOX gene is upregulated by various stresses including H2 O2 , heat, high light illumination, and iron or nitrogen limitation. AOX knockdown results in hypersensitivity to stress. Knockdown lines also show significantly reduced photosynthetic rates and their chloroplasts are more oxidized. Comparisons of transcriptome and metabolome profiles suggest a strong impact of AOX activity on gene expression, which is carried through to the level of the metabolome. Our data provide evidence for the involvement of mitochondrial AOX in processes central to the cell biology of diatoms, revealing that cross-talk between mitochondria and chloroplasts is crucial for maintaining sensitivity to changing environments.

Recent advances in next generation sequencing technologies have allowed the discovery of widespread autosomal allele-specific expression (aASE) in mammals and plants with potential phenotypic effects. Extensive numbers of genes with allele-specific expression have been described in the diatom Fragilariopsis cylindrus in association with adaptation to external cues, as well as in Fistulifera solaris in the context of natural hybridization. However, the role of aASE and its extent in diatoms remain elusive. In this study, we investigate allele-specific expression in the model diatom Phaeodactylum tricornutum by the re-analysis of previously published whole genome RNA sequencing data and polymorphism calling. We found that 22% of P. tricornutum genes show moderate bias in allelic expression while 1% show nearly complete monoallelic expression. Biallelic expression associates with genes encoding components of protein metabolism while moderately biased genes associate with functions in catabolism and protein transport. We validated candidate genes by pyrosequencing and found that moderate biases in allelic expression were less stable than monoallelically expressed genes that showed consistent bias upon experimental validations at the population level and in subcloning experiments. Our approach provides the basis for the analysis of aASE in P. tricornutum and could be routinely implemented to test for variations in allele expression under different environmental conditions.

SummaryIn multicellular organisms, Polycomb Repressive Complex2 (PRC2) is known to deposit H3K27me3 to establish and maintain gene silencing, critical for developmentally regulated processes. PRC2 complex is absent in both widely studied model yeasts which initially suggested that PRC2 arose with the emergence of multicellularity. However, its discovery in several unicellular species including microalgae questions its role in unicellular eukaryotes.Here, we use Phaeodactylum tricornutum enhancer of zeste E(z) knockouts and show that P. tricornutum E(z) is responsible for di and tri-methylation of lysine 27 of histone H3.H3K27me3 depletion abolishes cell morphology in P. tricornutum providing evidence for its role in cell differentiation. Genome wide profiling of H3K27me3 in fusiform and triradiate cells further revealed genes that may specify cell identity.These results suggest a role for PRC2 and its associated mark in cell differentiation in unicellular species and highlight their ancestral function in a broader evolutionary context than is currently appreciated.

SummaryIn many eukaryotes, such as dioicous mosses and many algae, sex is determined by UV sex chromosomes and is expressed during the haploid phase of the life cycle. In these species, the male and female developmental programs are initiated by the presence of the U- or V-specific regions of the sex chromosomes but, as in XY and ZW systems, phenotypic differentiation is largely driven by autosomal sex-biased gene expression. The mechanisms underlying sex-biased transcription in XY, ZW or UV sexual systems currently remain elusive. Here, we set out to understand the extent and nature of epigenomic changes associated with sexual differentiation in the brown alga Ectocarpus, which has a well described UV system. Five histone modifications, H3K4me3, H3K27Ac, H3K9Ac, H3K36me3, H4K20me3, were quantified in near-isogenic male and female lines, leading to the identification of 13 different chromatin states across the Ectocarpus genome that showed different patterns of enrichment at transcribed, silent, housekeeping or narrowly-expressed genes. Chromatin states were strongly correlated with levels of gene expression indicating a relationship between the assayed marks and gene transcription. The relative proportion of each chromatin state across the genome remained stable in males and females, but a subset of genes exhibited different chromatin states in the two sexes. In particular, males and females displayed distinct patterns of histone modifications at sex-biased genes, indicating that chromatin state transitions occur preferentially at genes involved in sex-specific pathways. Finally, our results reveal a unique chromatin landscape of the U and V sex chromosomes compared to autosomes. Taken together, our observations reveal a role for histone modifications in sex determination and sexual differentiation in a UV sexual system, and suggest that the mechanisms of epigenetic regulation of genes on the UV sex chromosomes may differ from those operating on autosomal genes.

International audience; Transcriptional coordination is a fundamental component of prokaryotic and eukaryotic cell biology, underpinning the cell cycle, physiological transitions, and facilitating holistic responses to environmental stress, but its overall dynamics in eukaryotic algae remain poorly understood. Better understanding of transcriptional partitioning may provide key insights into the primary metabolism pathways of eukaryotic algae, which frequently depend on intricate metabolic associations between the chloroplasts and mitochondria that are not found in plants. Here, we exploit 187 publically available RNAseq datasets generated under varying nitrogen, iron and phosphate growth conditions to understand the co-regulatory principles underpinning transcription in the model diatom Phaeodactylum tricornutum. Using WGCNA (Weighted Gene Correlation Network Analysis), we identify 28 merged modules of co-expressed genes in the P. tricornutum genome, which show high connectivity and correlate well with previous microarray-based surveys of gene co-regulation in this species. We use combined functional, subcellular localization and evolutionary annotations to reveal the fundamental principles underpinning the transcriptional co-regulation of genes implicated in P. tricornutum chloroplast and mitochondrial metabolism, as well as the functions of diverse transcription factors underpinning this co-regulation. The resource is publically available as PhaeoNet, an advanced tool to understand diatom gene co-regulation.

Diatoms are an ecologically fundamental and highly diverse group of algae, dominating marine primary production in both open-water and coastal communities. The diatoms include both centric species, which may have radial or polar symmetry, and the pennates, which include raphid and araphid species and arose within the centric lineage. Here, we use combined microscopic and molecular information to reclassify a diatom strain CCMP470, previously annotated as a radial centric species related to Leptocylindrus danicus, as an araphid pennate species in the staurosiroid lineage, within the genus Plagiostriata. CCMP470 shares key ultrastructural features with Plagiostriata taxa, such as the presence of a sternum with parallel striae, and the presence of a highly reduced labiate process on its valve; and this evolutionary position is robustly supported by multigene phylogenetic analysis. We additionally present a draft genome of CCMP470, which is the first genome available for a staurosiroid lineage. 270 Pfams (19%) found in the CCMP470 genome are not known in other diatom genomes, which otherwise does not hold big novelties compared to genomes of non-staurosiroid diatoms. Notably, our DNA library contains the genome of a bacterium within the Rhodobacterales, an alpha-proteobacterial lineage known frequently to associate with algae. We demonstrate the presence of commensal alpha-proteobacterial sequences in other published algal genome and transcriptome datasets, which may indicate widespread and persistent co-occurrence.

Background Brown algae evolved complex multicellularity independently of the animal and land plant lineages and are the third most developmentally complex phylogenetic group on the planet. An understanding of developmental processes in this group is expected to provide important insights into the evolutionary events necessary for the emergence of complex multicellularity. Here, we focus on mechanisms of epigenetic regulation involving post-translational modifications of histone proteins. Results A total of 47 histone post-translational modifications are identified, including a novel mark H2AZR38me1, but Ectocarpus lacks both H3K27me3 and the major polycomb complexes. ChIP-seq identifies modifications associated with transcription start sites and gene bodies of active genes and with transposons. H3K79me2 exhibits an unusual pattern, often marking large genomic regions spanning several genes. Transcription start sites of closely spaced, divergently transcribed gene pairs share a common nucleosome-depleted region and exhibit shared histone modification peaks. Overall, patterns of histone modifications are stable through the life cycle. Analysis of histone modifications at generation-biased genes identifies a correlation between the presence of specific chromatin marks and the level of gene expression. Conclusions The overview of histone post-translational modifications in the brown alga presented here will provide a foundation for future studies aimed at understanding the role of chromatin modifications in the regulation of brown algal genomes.

Diatoms are one of the most diverse and successful groups of phytoplankton at the base of the food chain, sustaining life in the ocean and performing vital biogeochemical functions. The last fifteen years have witnessed the comprehensive analysis of several diatom genomes, revealing that they bear traces of their endosymbiotic origins from algal and heterotrophic ancestors, as well as significant gene transfer from bacteria. Their chimeric genomes are further regulated by a range of chromatin-based processes that are characteristic of both plant and animal genomes. We discuss the conservation of gene regulatory mechanisms in diatoms and propose that epigenetic processes may have a significant role in mediating responses to a highly dynamic and unpredictable environment in these organisms.

Brown algae have convergently evolved plant-like body plans and reproductive cycles, which in plants are controlled by differential DNA methylation. This contribution provides the first single-base methylome profiles of haploid gametophytes and diploid sporophytes of a multicellular alga. Although only c. 1.4% of cytosines in Saccharina japonica were methylated mainly at CHH sites and characterized by 5-methylcytosine (5mC), there were significant differences between life-cycle stages. DNA methyltransferase 2 (DNMT2), known to efficiently catalyze tRNA methylation, is assumed to methylate the genome of S.japonica in the structural context of tRNAs as the genome does not encode any other DNA methyltransferases. Circular and long noncoding RNA genes were the most strongly methylated regulatory elements in S.japonica. Differential expression of genes was negatively correlated with DNA methylation with the highest methylation levels measured in both haploid gametophytes. Hypomethylated and highly expressed genes in diploid sporophytes included genes involved in morphogenesis and halogen metabolism. The data herein provide evidence that cytosine methylation, although occurring at a low level, is significantly contributing to the formation of different life-cycle stages, tissue differentiation and metabolism in brown algae.

Understanding of the molecular responses underpinning diatom responses to ocean acidification is fundamental for predicting how important primary producers will be shaped by the continuous rise in atmospheric CO2. In this study, we have analyzed global transcriptomic changes of the model diatom Phaeodactylum tricornutum following growth for 15 generations in elevated pCO2 by strand-specific RNA sequencing (ssRNA-seq). Our results indicate that no significant effects of elevated pCO2 and associated carbonate chemistry changes on the physiological performance of the cells were observed after 15 generations whereas the expression of genes encoding histones and other genes involved in chromatin structure were significantly down-regulated, while the expression of transposable elements (TEs) and genes encoding histone acetylation enzymes were significantly up-regulated. Furthermore, we identified a series of long non-protein coding RNAs (lncRNAs) specifically responsive to elevated pCO2, suggesting putative regulatory roles for these largely uncharacterized genome components. Taken together, our integrative analyses reveal that epigenetic elements such as TEs, histone modifications and lncRNAs may have important roles in the acclimation of diatoms to elevated pCO2 over short time scales and thus may influence longer term adaptive processes in response to progressive ocean acidification.

The marine pennate diatom Phaeodactylum tricornutum has become a model for diatom biology, due to its ease of culture and accessibility to reverse genetics approaches. While several features underlying the molecular mechanisms of cell division have been described, morphological analyses are less advanced than they are in other diatoms. We therefore examined cell ultrastructure changes prior to and during cytokinesis. Following chloroplast division, cleavage furrows are formed at both longitudinal ends of the cell and are accompanied by significant vesicle transport. Although neither spindle nor microtubules were observed, the nucleus appeared to be split by the furrow after duplication of the Golgi apparatus. Finally, centripetal cytokinesis was completed by fusion of the furrows. Additionally, F-actin formed a ring structure and its diameter became smaller, accompanying the ingrowing furrows. To further analyse vesicular transport during cytokinesis, we generated transgenic cells expressing yellow fluorescent protein (YFP) fusions with putative diatom orthologs of small GTPase Sec4 and t-SNARE protein SyntaxinA. Time-lapse observations revealed that SyntaxinA-YFP localization expands from both cell tips toward the center, whereas Sec4-YFP was found in the Golgi and subsequently relocalizes to the future division plane. This work provides fundamental new information about cell replication processes in P. tricornutum.

Background Allopolyploidy is a genomic structure wherein two or more sets of chromosomes derived from divergent parental species coexist within an organism. It is a prevalent genomic configuration in plants, as an important source of genetic variation, and also frequently confers environmental adaptability and increased crop productivity. We previously reported the oleaginous marine diatom Fistulifera solaris JPCC DA0580 to be a promising host for biofuel production and that its genome is allopolyploid, which had never previously been reported in eukaryotic microalgae. However, the study of allopolyploidy in F. solaris was hindered by the difficulty in classifying the homoeologous genes based on their progenitor origins, owing to the shortage of diatom genomic references. Results In this study, the allopolyploid genome of F. solaris was tentatively classified into two pseudo-parental subgenomes using sequence analysis based on GC content and codon frequency in each homoeologous gene pair. This approach clearly separated the genome into two distinct fractions, subgenome Fso_h and Fso_l, which also showed the potency of codon usage analysis to differentiate the allopolyploid subgenome. Subsequent homoeolog expression bias analysis revealed that, although both subgenomes appear to contribute to global transcription, there were subgenomic preferences in approximately 61% of homoeologous gene pairs, and the majority of these genes showed continuous bias towards a specific subgenome during lipid accumulation. Additional promoter analysis indicated the possibility of promoter motifs involved in biased transcription of homoeologous genes. Among these subgenomic preferences, genes involved in lipid metabolic pathways showed interesting patterns in that biosynthetic and degradative pathways showed opposite subgenomic preferences, suggesting the possibility that the oleaginous characteristics of F. solaris derived from one of its progenitors. Conclusions We report the detailed genomic structure and expression patterns in the allopolyploid eukaryotic microalga F. solaris. The allele-specific patterns reported may contribute to the oleaginous characteristics of F. solaris and also suggest the robust oleaginous characteristics of one of its progenitors. Our data reveal novel aspects of allopolyploidy in a diatom that is not only important for evolutionary studies but may also be advantageous for biofuel production in microalgae. Electronic supplementary material The online version of this article (10.1186/s12864-018-4691-0) contains supplementary material, which is available to authorized users.

While our knowledge about the roles of microbes and viruses in the ocean has increased tremendously due to recent advances in genomics and metagenomics, research on marine microbial eukaryotes and zooplankton has benefited much less from these new technologies because of their larger genomes, their enormous diversity, and largely unexplored physiologies. Here, we use a metatranscriptomics approach to capture expressed genes in open ocean Tara Oceans stations across four organismal size fractions. The individual sequence reads cluster into 116 million unigenes representing the largest reference collection of eukaryotic transcripts from any single biome. The catalog is used to unveil functions expressed by eukaryotic marine plankton, and to assess their functional biogeography. Almost half of the sequences have no similarity with known proteins, and a great number belong to new gene families with a restricted distribution in the ocean. Overall, the resource provides the foundations for exploring the roles of marine eukaryotes in ocean ecology and biogeochemistry., Marine microbial eukaryotes and zooplankton display enormous diversity and largely unexplored physiologies. Here, the authors use metatranscriptomics to analyze four organismal size fractions from open-ocean stations, providing the largest reference collection of eukaryotic transcripts from any single biome.

Recent progress made on epigenetic studies revealed the conservation of epigenetic features in deep diverse branching species including Stramenopiles, plants and animals. This suggests their fundamental role in shaping species genomes across different evolutionary time scales. Diatoms are a highly successful and diverse group of phytoplankton with a fossil record of about 190 million years ago. They are distantly related from other super-groups of Eukaryotes and have retained some of the epigenetic features found in mammals and plants suggesting their ancient origin. Phaeodactylum tricornutum and Thalassiosira pseudonana, pennate and centric diatoms, respectively, emerged as model species to address questions on the evolution of epigenetic phenomena such as what has been lost, retained or has evolved in contemporary species. In the present work, we will discuss how the study of non-model or emerging model organisms, such as diatoms, helps understand the evolutionary history of epigenetic mechanisms with a particular focus on DNA methylation and histone modifications.

Diatoms are one of the most successful and ecologically important groups of eukaryotic phytoplankton in the modern ocean. Deciphering their genomes is a key step towards better understanding of their biological innovations, evolutionary origins, and ecological underpinnings. Here, we have used 90 RNA-Seq datasets from different growth conditions combined with published expressed sequence tags and protein sequences from multiple taxa to explore the genome of the model diatomPhaeodactylum tricornutum,and introduce 1,489 novel genes. The new annotation additionally permitted the discovery for the first time of extensive alternative splicing (AS) in diatoms, including intron retention and exon skipping which increases the diversity of transcripts to regulate gene expression in response to nutrient limitations. In addition, we have used up-to-date reference sequence libraries to dissect the taxonomic origins of diatom genomes. We show that theP. tricornutumgenome is replete in lineage-specific genes, with up to 47% of the gene models present only possessing orthologues in other stramenopile groups. Finally, we have performed a comprehensivede novoannotation of repetitive elements showing novel classes of TEs such as SINE, MITE, LINE and TRIM/LARD. This work provides a solid foundation for future studies of diatom gene function, evolution and ecology.

Diatoms emerged in the Mesozoic period and presently constitute one of the main primary producers in the world’s ocean and are of a major economic importance. In the current study, using whole genome sequencing of ten accessions of the model diatomPhaeodactylum tricornutum, sampled at broad geospatial and temporal scales, we draw a comprehensive landscape of the genomic diversity within the species. We describe strong genetic subdivisions of the accessions into four genetic clades (A-D) with constituent populations of each clade possessing a conserved genetic and functional makeup, likely a consequence of the limited dispersal ofP. tricornutumin the open ocean. We further suggest dominance of asexual reproduction across all the populations, as implied by high linkage disequilibrium. Finally, we show limited yet compelling signatures of genetic and functional convergence inducing changes in the selection pressure on many genes and metabolic pathways. We propose these findings to have significant implications for understanding the genetic structure of diatom populations in nature and provide a framework to assess the genomic underpinnings of their ecological success and impact on aquatic ecosystems where they play a major role. Our work provides valuable resources for functional genomics and for exploiting the biotechnological potential of this model diatom species.

Background With the emerging interest in phytoplankton research, the need to establish genetic tools for the functional characterization of genes is indispensable. The CRISPR/Cas9 system is now well recognized as an efficient and accurate reverse genetic tool for genome editing. Several computational tools have been published allowing researchers to find candidate target sequences for the engineering of the CRISPR vectors, while searching possible off-targets for the predicted candidates. These tools provide built-in genome databases of common model organisms that are used for CRISPR target prediction. Although their predictions are highly sensitive, the applicability to non-model genomes, most notably protists, makes their design inadequate. This motivated us to design a new CRISPR target finding tool, PhytoCRISP-Ex. Our software offers CRIPSR target predictions using an extended list of phytoplankton genomes and also delivers a user-friendly standalone application that can be used for any genome. Results The software attempts to integrate, for the first time, most available phytoplankton genomes information and provide a web-based platform for Cas9 target prediction within them with high sensitivity. By offering a standalone version, PhytoCRISP-Ex maintains an independence to be used with any organism and widens its applicability in high throughput pipelines. PhytoCRISP-Ex out pars all the existing tools by computing the availability of restriction sites over the most probable Cas9 cleavage sites, which can be ideal for mutant screens. Conclusions PhytoCRISP-Ex is a simple, fast and accurate web interface with 13 pre-indexed and presently updating phytoplankton genomes. The software was also designed as a UNIX-based standalone application that allows the user to search for target sequences in the genomes of a variety of other species. Electronic supplementary material The online version of this article (doi:10.1186/s12859-016-1143-1) contains supplementary material, which is available to authorized users.

The last decade has witnessed outstanding progress in sequencing the genomes of photosynthetic eukaryotes, from major cereal crops to single celled marine phytoplankton. For the algae, we now have whole genome sequences from green, red, and brown representatives, and multiple efforts based on comparative and functional genomics approaches have provided information about the unicellular origins of higher plants, and about the evolution of photosynthetic life in general. Here we present some of the highlights from such studies, including the endosymbiotic origins of photosynthetic protists and their positioning with respect to plants and animals, the evolution of multicellularity in photosynthetic lineages, the role of sex in unicellular algae, and the potential relevance of epigenetic processes in contributing to the adaptation of algae to their environment.

We have identified an active Medicago truncatula copia-like retroelement called Medicago RetroElement1-1 (MERE1-1) as an insertion in the symbiotic NSP2 gene. MERE1-1 belongs to a low-copy-number family in the sequenced Medicago genome. These copies are highly related, but only three of them have a complete coding region and polymorphism exists between the long terminal repeats of these different copies. This retroelement family is present in all M. truncatula ecotypes tested but also in other legume species like Lotus japonicus. It is active only during tissue culture in both R108 and Jemalong Medicago accessions and inserts preferentially in genes.

Legume root nodules originate from differentiated cortical cells that reenter the cell cycle and form organ primordia. We show that perception of the phytohormone cytokinin is a key element in this switch. Mutation of a Lotus japonicus cytokinin receptor gene leads to spontaneous development of root nodules in the absence of rhizobia or rhizobial signal molecules. The mutant histidine kinase receptor has cytokinin-independent activity and activates an Escherichia coli two-component phosphorelay system in vivo. Mutant analysis shows that cytokinin signaling is required for cell divisions that initiate nodule development and defines an autoregulated process where cytokinin induction of nodule stem cells is controlled by shoots.

International audience; Diatoms are one of the most ecologically successful classes of photosynthetic marine eukaryotes in the contemporary oceans. Over the past 30 million years, they have helped to moderate Earth's climate by absorbing carbon dioxide from the atmosphere, sequestering it via the biological carbon pump and ultimately burying organic carbon in the lithosphere. The proportion of planetary primary production by diatoms in the modern oceans is roughly equivalent to that of terrestrial rainforests. In photosynthesis, the efficient conversion of carbon dioxide into organic matter requires a tight control of the ATP/NADPH ratio which, in other photosynthetic organisms, relies principally on a range of plastid-localized ATP generating processes. Here we show that diatoms regulate ATP/NADPH through extensive energetic exchanges between plastids and mitochondria. This interaction comprises the re-routing of reducing power generated in the plastid towards mitochondria and the import of mitochondrial ATP into the plastid, and is mandatory for optimized carbon fixation and growth. We propose that the process may have contributed to the ecological success of diatoms in the ocean.

Background: Nucleosomes are the building blocks of chromatin where gene regulation takes place. Chromatin landscapes have been profiled for several species, providing insights into the fundamental mechanisms of chromatin-mediated transcriptional regulation of gene expression. However, knowledge is missing for several major and deep-branching eukaryotic groups, such as the Stramenopiles, which include the diatoms. Diatoms are highly diverse and ubiquitous species of phytoplankton that play a key role in global biogeochemical cycles. Dissecting chromatin-mediated regulation of genes in diatoms will help understand the ecological success of these organisms in contemporary oceans. Results: Here, we use high resolution mass spectrometry to identify a full repertoire of post-translational modifications on histones of the marine diatom Phaeodactylum tricornutum, including eight novel modifications. We map five histone marks coupled with expression data and show that P. tricornutum displays both unique and broadly conserved chromatin features, reflecting the chimeric nature of its genome. Combinatorial analysis of histone marks and DNA methylation demonstrates the presence of an epigenetic code defining activating or repressive chromatin states. We further profile three specific histone marks under conditions of nitrate depletion and show that the histone code is dynamic and targets specific sets of genes. Conclusions: This study is the first genome-wide characterization of the histone code from a stramenopile and a marine phytoplankton. The work represents an important initial step for understanding the evolutionary history of chromatin and how epigenetic modifications affect gene expression in response to environmental cues in marine environments. Instituto de Biotecnología Fil: Veluchamy, Alaguraj. Centre National de la Recherche Scientifique. Institut de Biologie de l’École Normale Supérieure. Ecology and Evolutionary Biology Section; Francia. King Abdullah University of Science and Technology. BESE Division. Center for Desert Agriculture; Arabia Saudita Fil: Rastogi, Achal. Centre National de la Recherche Scientifique. Institut de Biologie de l’École Normale Supérieure. Ecology and Evolutionary Biology Section; Francia Fil: Lin, Xin. Centre National de la Recherche Scientifique. Institut de Biologie de l’École Normale Supérieure. Ecology and Evolutionary Biology Section; Francia. Xiamen University. State key lab of Marine Environmental Science; China Fil: Lombard, Bérangère. Institut Curie PSL Research University. Centre de Recherche. Laboratoire de Spectrométrie de Masse Protéomique; Francia Fil: Murik, Omer. Centre National de la Recherche Scientifique. Institut de Biologie de l’École Normale Supérieure. Ecology and Evolutionary Biology Section; Francia Fil: Thomas, Yann. Centre National de la Recherche Scientifique. Institut de Biologie de l’École Normale Supérieure. Ecology and Evolutionary Biology Section; Francia Fil: Dingli, Florent. Institut Curie PSL Research University. Centre de Recherche. Laboratoire de Spectrométrie de Masse Protéomique; Francia Fil: Rivarola, Maximo Lisandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. University of Maryland School of Medicine. Institute for Genome Sciences; Estados Unidos Fil: Ott, Sandra. University of Maryland School of Medicine. Institute for Genome Sciences; Estados Unidos Fil: Liu, Xinyue. University of Maryland School of Medicine. Institute for Genome Sciences; Estados Unidos Fil: Sun, Yezhou. University of Maryland School of Medicine. Institute for Genome Sciences; Estados Unidos Fil: Rabinowicz, Pablo D. University of Maryland School of Medicine. Institute for Genome Sciences; Estados Unidos Fil: McCarthy, James. J. Craig Venter Institute; Estados Unidos Fil: Allen, Andrew E. J. Craig Venter Institute; Estados Unidos. University of California. Integrative Oceanography Division. Scripps Institution of Oceanography; Estados Unidos Fil: Loew, Damarys. Institut Curie PSL Research University. Centre de Recherche. Laboratoire de Spectrométrie de Masse Protéomique; Francia Fil: Bowler, Chris. Centre National de la Recherche Scientifique. Institut de Biologie de l’École Normale Supérieure. Ecology and Evolutionary Biology Section; Francia Fil: Tirichine, Leïla. Centre National de la Recherche Scientifique. Institut de Biologie de l’École Normale Supérieure. Ecology and Evolutionary Biology Section; Francia

International audience; Numerous cellular functions including respiration require iron. Plants and phytoplankton must also maintain the iron-rich photosynthetic electron transport chain, which most likely evolved in the iron-replete reducing environments of the Proterozoic ocean [1]. Iron bioavailability has drastically decreased in the contemporary ocean [1], most likely selecting for the evolution of efficient iron acquisition mechanisms among modern phytoplankton. Mesoscale iron fertilization experiments often result in blooms dominated by diatoms [2], indicating that diatoms have adaptations that allow survival in iron-limited waters and rapid multiplication when iron becomes available. Yet the genetic and molecular bases are unclear, as very few iron uptake genes have been functionally characterized from marine eukaryotic phytoplankton, and large portions of diatom iron starvation transcriptomes are genes encoding unknown functions [3-5]. Here we show that the marine diatom Phaeodactylum tricornutum utilizes ISIP2a to concentrate Fe(III) at the cell surface as part of a novel, copper-independent and thermodynamically controlled iron uptake system. ISIP2a is expressed in response to iron limitation several days prior to the induction of ferrireductase activity, and it facilitates significant Fe(III) uptake during the initial response to Fe limitation. ISIP2a is able to directly bind Fe(III) and increase iron uptake when heterologously expressed, whereas knockdown of ISIP2a in P. tricornutum decreases iron uptake, resulting in impaired growth and chlorosis during iron limitation. ISIP2a is expressed by diverse marine phytoplankton, indicating that it is an ecologically significant adaptation to the unique nutrient composition of marine environments.

International audience; Diatoms constitute a major phylum of phytoplankton biodiversity in ocean water and freshwater ecosystems. They are known to respond to some chemical variations of the environment by the accumulation of triacylglycerol, but the relative changes occurring in membrane glycerolipids have not yet been studied. Our goal was first to define a reference for the glycerolipidome of the marine model diatom Phaeodactylum tricornutum, a necessary prerequisite to characterize and dissect the lipid metabolic routes that are orchestrated and regulated to build up each subcellular membrane compartment. By combining multiple analytical techniques, we determined the glycerolipid profile of P. tricornutum grown with various levels of nitrogen or phosphorus supplies. In different P. tricornutum accessions collected worldwide, a deprivation of either nutrient triggered an accumulation of triacylglycerol, but with different time scales and magnitudes. We investigated in depth the effect of nutrient starvation on the Pt1 strain (Culture Collection of Algae and Protozoa no. 1055/3). Nitrogen deprivation was the more severe stress, triggering thylakoid senescence and growth arrest. By contrast, phosphorus deprivation induced a stepwise adaptive response. The time scale of the glycerolipidome changes and the comparison with large-scale transcriptome studies were consistent with an exhaustion of unknown primary phosphorus-storage molecules (possibly polyphosphate) and a transcriptional control of some genes coding for specific lipid synthesis enzymes. We propose that phospholipids are secondary phosphorus-storage molecules broken down upon phosphorus deprivation, while nonphosphorus lipids are synthesized consistently with a phosphatidylglycerol-to-sulfolipid and a phosphatidycholine-to-betaine lipid replacement followed by a late accumulation of triacylglycerol.

Corrigendum: Insights into the role of DNA methylation in diatoms by genome-wide profiling in Phaeodactylum tricornutum

The availability of a wide range of independent lines for the annual medic Medicago truncatula led us to search for natural variants in the symbiotic association with Sinorhizobium meliloti. Two homozygous lines, Jemalong 6 and DZA315.16, originating from an Australian cultivar and a natural Algerian population, respectively, were inoculated with two wild-type strains ofS. meliloti, RCR2011 and A145. Both plant lines formed nitrogen-fixing (effective) nodules with the RCR2011 strain. However, the A145 strain revealed a nitrogen fixation polymorphism, establishing an effective symbiosis (Nod+Fix+) with DZA315.16, whereas only small, white, non-nitrogen fixing nodules (Nod+Fix−) were elicited on Jemalong 6. Cytological studies demonstrated that these non-fixing nodules are encircled by an endodermis at late stages of development, with no visible meristem, and contain hypertrophied and autofluorescent infection threads, suggesting the induction of plant defense reactions. The non-fixing phenotype is independent of growth conditions and determined by a single recessive allele (Mtsym6), which is located on linkage group 8.