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Recent progress in diatom genomics and epigenomics
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ScienceDirect Recent progress in diatom genomics and epigenomics Leila Tirichine, Achal Rastogi and Chris Bowler 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.
Address Ecole Normale Supe´rieure, PSL Research University, Institut de Biologie de l’Ecole Normale Supe´rieure (IBENS), CNRS UMR 8197, INSERM U1024, 46 rue d’Ulm, F-75005 Paris, France Corresponding author: Tirichine, Leila ([email protected])
Current Opinion in Plant Biology 2017, 36:46–55 This review comes from a themed issue on Genome studies and molecular genetics Edited by Ian Henderson and Korbinian Schneeberger
http://dx.doi.org/10.1016/j.pbi.2017.02.001 1369-5266/ã 2017 Elsevier Ltd. All rights reserved.
Introduction Diatoms are highly diverse and cosmopolitan single celled eukaryotes of the Stramenopile lineage found in nearly all aquatic habitats including fresh and marine waters and moist soil. Diatoms date back to at least the early Jurassic (185 Ma ago), they survived the endCretaceous mass extinction relatively unscathed, and underwent a major diversification during the Cenozoic [1] that led to them becoming one of the major groups of phytoplankton in contemporary oceans [2]. They are believed to be derived from a serial secondary endosymbiosis approximately 1 billion years ago [3,4] in which a heterotrophic eukaryotic host domesticated photosynthetic eukaryotic cells. Current Opinion in Plant Biology 2017, 36:46–55
Like higher plants, diatoms are obligately photosynthetic. Current estimates indicate that they contribute around 40% of primary production in the ocean and 20% of global carbon fixation [5]. They are therefore a major component providing organic carbon to higher trophic levels in marine food webs, as well as participating in the global carbon cycle. Another feature of diatoms is their elaborate cell wall known as the frustule, composed of amorphous silica, which influences multiple critical aspects of diatom life histories, including cell division [6,7], susceptibility to grazing [8], and sinking rates from sunlit upper layers to the ocean interior. The latter process contributes to carbon export and is an extremely important biological process that removes carbon from the global carbon cycle over geological time scales [9], ultimately contributing to the generation of oil and gas fossil fuel reserves. Furthermore, the mineralization of silicon by diatoms is a major component of the silica biogeochemical cycle [10]. Fossilized frustules are valuable paleo tracers for geologists [11], and have accumulated in vast deposits of diatomaceous earth that are quarried for a range of applications [12–14]. Diatom frustules show a wide diversity of morphologies (Figure 1), which have been the basis of traditional diatom taxonomy, dividing them into two orders: centric diatoms that have radial symmetry and pennate diatoms that are elongate with thin ellipses. Centric diatoms are further divided into polar centrics (Mediophyceae) and radial centrics (Coscinodiscophyceae), and pennate diatoms into raphid (Bacillariophyceae) and araphid diatoms (Fragilariophyceae) which is based on the presence or absence of a raphe in the valve, which facilitates movement. Diatoms are also of interest for several applications including the production of therapeutics, biofuels and biopolymers [15,16]. They are increasingly being used as molecular farms to produce several molecules such as polyunsaturated fatty acids, carotenoids, polysaccharides, vitamins, and sterols [17], as well as vaccines and their adjuvants [18,19]. Today, materials from diatoms provide numerous items ranging from toothpaste, nail polish, filters for swimming pools and fountains, building materials, biofuel and diatomaceous earth for organic pest control. The ecological, evolutionary and biotechnological interests of diatoms have attracted the attention of the scientific community, leading to a significant increase in published articles that have led to tangible advances in knowledge, particularly enabled by genomics. Here we www.sciencedirect.com
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Figure 1
The morphological diversity of diatoms revealed by different microscopy techniques. Chain of pennate diatoms (top left) and individual centric diatom (top right) observed by epifluorescence microscopy. Cell impermeable Alexa Fluor 546 conjugates reveal extracellular structures (green), red chlorophyll autofluorescence reveals chloroplasts, and DAPI stains the nucleus in blue (only in top left panel). The image in top right is of Planktoniella, an open ocean genus characterized by an elaborate spoke-like extracellular structure that may facilitate buoyancy or provide protection from grazers. The lower two panels show scanning electron micrographs of the nanostructured silicified frustules of a pennate diatom (Fragilariopsis doliolus) (bottom left) and a centric diatom (possibly Thalassiosira) (bottom right). The diatoms were collected during the Tara Oceans expedition and images were made by Atsuko Tanaka. Scale bars are indicated.
present an overview of recent progress in diatom genomics, comparative genomics and epigenomics. We particularly review the different components of chromatinlevel regulation and highlight examples showing their contribution to genome regulation. Such examples are placed in the context of a chimeric genome derived from the unusual evolutionary history of diatoms that has brought together genes of red and green algal origin as well as from bacteria [20,21,22]. Allele specific expression is an additional feature that may permit further phenotypic plasticity and thus help diatoms to thrive in dynamic highly unstable environments.
Tiny but complex Thalassiosira pseudonana and Phaeodactylum tricornutum were the first diatom genomes to be sequenced, www.sciencedirect.com
representing centric and pennate diatoms, respectively [20,22]. Both species are on the small side for diatoms (less than 10 mm), as are their genomes, on the order of 30 Mb. Nonetheless, they each encode a plethora of genes, around 12 000, encoding diverse and complex functions likely acquired through their evolutionary history combining genes from exosymbiont as well as from algal endosymbionts and bacteria [20,21,22,23]. The unusual gene repertoire of diatoms has been proposed to underpin their ecological success in the contemporary ocean [24,25]. For example, diatom genomes have genes encoding a metazoan-like ornithine urea cycle that is absent from plants [20,26,27], large numbers of diatom-specific cyclins, heat shock transcription factors [7,28], and far-red light sensors related to phytochromes [29]. Current Opinion in Plant Biology 2017, 36:46–55
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Diatom genomes were furthermore shown to be compact with small gene sizes and intergenic regions, and a significant proportion of transposable elements (TEs) which predominantly belong to class I TEs, in particular the LTR retrotransposon superfamily [30]. Examination of LTR retro-elements of the copia type in both P. tricornutum and T. pseudonana revealed the existence of seven groups of diatom-specific TEs named CoDi (Copia-like elements from diatoms) [30]. Some of the CoDi groups were shown to be expressed under specific conditions in P. tricornutum and T. pseudonana suggesting a role of TEs in adaptation and diversification of diatoms [25,30]. Although challenging because of their repetitive sequences and dynamic behavior within a genome, TE annotation should be further pursued in diatoms to better understand their influence in diatom evolution, speciation, and adaptation to the environment.
Diatom eclectic diversity More than ten diatom whole genome sequences including the first freshwater diatom [31] are today available or being generated (Table 1). These genome sequences have been further enriched with transcriptomes from the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) [32] which has put together over 650 assembled, functionally annotated transcriptomes amongst which 92 diatom species (Figure 2, Table S1). From the most abundant diatom genera in the Tara Oceans global ocean plankton sampling survey [33], seven additional diatom transcriptomes have been generated by Genoscope (from Shionodiscus, Synedra, Pleurosigma, Navicula, Haslea, Guinardia and Minidiscus) (Table 1). This growing database of diatom sequences represents a valuable resource for functional and comparative genomics studies that can help understand
evolutionary histories and reveal the metabolic peculiarities that distinguish them from other species [26,34–36]. Genomics studies have so far examined only a tiny portion of the existing diversity of diatoms. A metabarcoding approach based on 18s rDNA sequences obtained from environmental samples collected from major oceanic provinces during the Tara Oceans expedition revealed 63 371 unique diatom-derived barcodes, potentially representing up to 4748 taxa [37], including a considerable number of abundant yet unidentifiable species. An important objective in future years will be to bring representatives of this unexplored diatom diversity into culture. The abundance of prokaryotes, eukaryotes and viruses at multiple sampling sites was further used to generate an ocean plankton interactome that revealed that collaboration rather than competition between organisms was more prevalent [38]. A conspicuous exception were the diatoms, that appear to exclude other organisms and thus to be more competitive than other phytoplankton groups [38]. Because diatoms (like other marine microbial microorganisms) are constituted by large populations with vast possibilities for geographic dispersal, rapid growth rates, and mainly asexual reproduction, they are classically considered as having a continuous distribution and weak biogeographic population structures [39]. However, the oceans are not homogeneous because of seascape topography and ocean circulation acting over multiple scales both horizontally and vertically, resulting in localized micro-environments that shape the diversity of the biota within them and create barriers to dispersal [40]. Indeed, changing conditions over time and space can lead to the appearance of specific endemic taxa that are able to
Table 1 Genomic features of the first sequenced diatom genomes. One important finding distinguishing pennate from centric diatoms is the fraction of genes (40%) that are not shared between the two lineages which can lead to differences such as the preference in codon usage in P. tricornutum that is different from T. pseudonana [36]. P. tricornutum seems to possess the smallest genome size as well as number of genes, the largest being the genome of Pseudo-nitzschia multiseries (Table 1). Some of the earliest sequenced genomes such as T. pseudonana and P. tricornutum have seen their annotation improved and several reiterations have been made since the first annotated draft. Phaeodactylum tricornutum annotation 3 (Phatr3) available at Ensembl revealed over 1000 new genes and transposable elements and improved significantly existing gene models (Rastogi et al., in review) Genome feature
Nuclear genome size (MB) Number of chromosome(s)/scaffold(s) Number of genes Average length of a gene (bp) Average length of an exon (bp) Average length of an intron (bp) Average protein length (aa) Average exon density per gene Current annotation version
Centric diatoms
Pennate diatoms
Thalassiosira pseudonana
Thalassiosira oceanica
Phaeodactylum tricornutum
Fragilariopsis cylindrus
Pseudo-nitzschia multiseries
32.1 65 11 776 1674 571 129 464 2.6 v3 (2007)
92.0 51 656 34 500 1256 464 146 354 2.3 v1 (2014)
27.4 88 12 177 1624 886 142 495 1.7 v3 (2016)
80.5 271 27 137 1572 615 256 409 2.1 v1 (2008)
218.73 4890 19 703 1522 509 229 377 2.4 v1 (2011)
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Figure 2
The evolutionary divergence of diatoms. Sequences from 57 diatom species, whose transcriptome sequences are available in the MMETSP database and 18S gene reference is available in PR2 database, were used to generate a phylogenetic tree using the Neighbor-Joining method [84]. The tree shows the phylogenetic association between the newly sequenced diatom species with model species such as P. tricornutum and T. pseudonana. The tree was generated using 18S rRNA gene sequences taken from the PR2 database [85]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [86]. The evolutionary distances were computed using the Maximum Composite Likelihood method [87] and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA7 [88]. Boxed in red color, the tree is rooted to Bolidophytes, sister lineage to diatoms. Node prefixed by black diamonds indicates pennate diatom species and others are centric diatoms. The tree is clustered into four subclasses of diatom, which is based on the valve organization of the diatom cell.
outcompete conspecifics and evolve into distinct species [41,42,43]. Furthermore, different diatom species can coexist within the same water column spanning a range of depths during a bloom suggesting that the ocean contains a pool of standing stock genetic diversity upon which selection can act [44]. Diatoms are known to display high diversification rates which have been proposed to be generated at least in part as a result of occasional sexual reproduction events between genetically distinguishable taxa, thus creating natural hybrids that can disperse diversity within a population [45]. The genome sequences of both Fistulifera solaris and Fragilariopsis cylindrus have provided further molecular evidence for such a phenomenon [16] (Mock et al., in press). For www.sciencedirect.com
example, in F. cylindrus 45% of divergent alleles showed an allele specific expression in at least one of the tested conditions, suggesting the importance of differential allele expression in fine tuning gene regulation in response to the environment (Mock et al., in press). It therefore seems possible that heterosis may be pervasive in natural diatom populations. Natural hybridization has been proposed previously to play a role in generating diversity within multiple marine micro-eukaryotes, including diatoms [16,46,47] and dinoflagellates [48,49], suggesting its potential role in creating heterogeneity and thus diversity within a species and/or genus. Bi- and mono-allelism, defined as the expression of either both alleles or only one allele in a diploid cell, has been Current Opinion in Plant Biology 2017, 36:46–55
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shown to occur in specific classes of genes in many organisms, expanding their diversity and adaptability, playing thus a significant role in their evolution [50]. This is typically the case for genes encoding olfactory and immune receptors in mammals, allowing progeny recognition and survival, and increasing disease resistance [51]. It will be of great interest to examine the extent of the phenomenon in diatoms, both in laboratory and natural conditions.
A growing toolbox to determine gene function in diatoms Sequencing diatom genomes and transcriptomes is one step towards decoding and understanding their genome, physiology, ecology, and evolutionary history. But another challenge is to determine the function of individual genes, in particular those that have no functional information assigned to them following in silico annotation. In a case study, the fact that a significant proportion of P. tricornutum genes (13%) are diatom-specific [20] (Rastogi et al., in revision) means that, in the overwhelming majority of cases, we have no functional information about the proteins encoded by them. With this in mind, over the last two decades the diatom community has developed tools and methods to explore gene function. Both P. tricornutum and T. pseudonana are now amenable to reverse genetics [52,53], although the superior tools developed for P. tricornutum have made it the experimental system of choice for most studies. Interest in using P. tricornutum as a cell factory for the production of highvalue products is facilitating this progress [54,55], as is the use of the species to explore diatom biology in an ecological context. Although found only in coastal areas and considered to be of limited ecological relevance, P. tricornutum has nonetheless demonstrated its usefulness in this regards because of the conservation of basic mechanisms among diatoms. This is illustrated by a study of iron uptake pathways in P. tricornutum which were shown to be similar to other low iron quota ecologically relevant diatoms, including pennate and centric species [35]. P. tricornutum is a robust laboratory species that can grow axenically in a range of culture media. It is less silicified compared to other diatoms, which is a considerable advantage for introducing exogenous DNA; in fact the species is amenable for transformation by microparticle gun bombardment [56], conjugation with Escherichia coli, and electroporation [57,58,59]. Different knock-down/ out strategies are feasible in P. tricornutum including RNA interference, TALEN, Artificial MicroRNA, and lately CRISPR cas9 editing [52,53,60,61]. Furthermore, digital gene expression approaches have been designed that permit the identification of candidate genes of potential importance for responding to a range of environmentallyrelevant signals [62]. Such examples include studies using RNAi silencing to characterize the peculiar urea cycle found in diatoms [26], the iron starvation induced Current Opinion in Plant Biology 2017, 36:46–55
protein ISIP2a, a protein that was shown to concentrate Fe(III) at the cell surface facilitating its uptake in iron limiting conditions [35], an alternative oxidase involved in extensive cross-talk between mitochondria and chloroplasts that improves photosynthetic efficiency [63], and diatom-specific cyclins implicated in the control of cell division by light [7,35,63,64]. More recently, several genes have been functionally characterized using knockout tools, including the far red light sensing DPH protein in P. tricornutum and T. pseudonana [29] which is surprising because of the absence or scarcity of such long wavelengths of light in subsurface waters. Altogether, these different studies indicate that the investigated genes contribute to the ecological success of diatoms in the ocean.
Conservation and surprises in the epigenetic machinery of diatoms In silico analyses of available diatom whole genome sequences have revealed the existence of nearly all components of the epigenetic machinery normally found in higher eukaryotes [65,66], suggesting the ancient origin of this mode of genome regulation. A striking example is cytosine methylation, which was first detected by HPLC and then molecularly using McrBC digestion coupled to microarray hybridization, and bisulfite sequencing [67]. The study by Veluchamy et al. found that in P. tricornutum a low percentage of DNA methylation can be found over around 5% of genes, intergenic regions, and TEs, and in all contexts (CG, CHH and CHG, where H can be any nucleotide except G) suggesting that this is not a plantspecific feature, as previously thought [67], but in fact evolved long before in eukaryote evolution. Later work found similar patterns of DNA methylation in both T. pseudonana, F. cylindrus and more recently in Cyclotella cryptica where 61% of the genome was found methylated, which is the highest methylation level currently known in a diatom species [68,69,70]. Furthermore, a dynamic regulation of DNA methylation was observed in response to nitrate starvation, with both genes and TEs being differentially methylated, suggesting a role in surviving nutrient limitation in the ocean [67]. Some DNA methylated regions (DMRs) are highly packed with small RNAs [71] suggesting an RNA-directed DNA methylation process which is further supported by in silico evidence of genes encoding DNA polymerases PolIV and V in P. tricornutum (Tirichine, unpublished). Non-coding RNAs have in fact been reported in diatoms by several groups although an in-depth functional characterization is still lacking [71–75]. Canonical miRNAs which have a typical complex for generating precursor miRNAs (Drosha) were not detected in the studied diatoms, but only 25–30 nt-long RNAs found to map principally to repeats, TEs and a subset of genes. Interestingly, some of these small RNAs display a 180 nt-long periodic distribution at several locations in the genome of P. tricornutum [71] suggesting a possible nucleosome-mediated www.sciencedirect.com
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distribution of sRNAs as described previously for DNA methylation [68]. The latter study showed a clustered methylation over nucleosome linkers contributing therefore to nucleosome positioning [68] between clusters which might also be mediated by small RNAs. This periodic methylation was identified in multiple species of Mamiellophyceae which might be the result of their small cell size and compact genome, features shared with diatoms, likely facilitating nuclear functions in extremely high DNA densities [68].
Furthermore, post translational modifications (PTMs) of histones were detected using mass spectrometry in both P. tricornutum and T. pseudonana [76,77]. An interesting finding is that P. tricornutum combines PTMs specific to plants or animals which might reflect the chimeric nature of its genome [77]. Whole genome mapping of a few key PTMs in P. tricornutum revealed a conserved histone code more similar to animals than plants [77] (Figure 3). This histone or epigenetic code implies that histone marks and DNA methylation co-occur to determine chromatin states
Figure 3
Chromatin features in P. tricornutum. Two snapshots of a 12 kb region (a) and 3 kb region (b) from P. tricornutum genome showing the distribution of five chromatin marks (tracks H3K4me2, H3K9/14Ac, H3K9me2, H3K9me3, and H3K27me3), nucleosome positioning (track H3; using anti-H3 antibody [77]), as well as DNA methylation (track DNA meth.) [67,68] over genes and transposable elements (TEs). These snapshots show the presence of a histone code that regulates gene expression, with genes marked by active marks while TEs are co-marked by repressive marks known to be mutually exclusive in other lineages such as plants. Repressive marks such as H3K27me3, H3K9me2/3 and DNA methylation co-localize on TEs and sometimes on genes (b) which lead to the repression of their expression (indicated by reduced RPKM values) [77]. Note spreading of H3K9me2 to the neighboring gene which causes a diminution in its expression. The active marks H3K4me2 and H3K9/ 14Ac co-localize on genes that are actively transcribed. www.sciencedirect.com
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that are either repressive or active, leading to expression or repression of genes and transposable elements. One striking pattern is that of H3K27me3, a mark deposited by polycomb protein complex 2 (PRC2) which targets mainly TEs and only 10% of genes [77]. This is different from plants such as Arabidopsis where the mark covers only genes but is similar to animals and the filamentous fungus Neurospora crassa, suggesting similar roles in defining a facultative heterochromatin [77]. The same study revealed another interesting feature which is the cooccurrence of H3K27me3 with two other repressive marks (H3K9me2/me3), as well as DNA methylation over TEs and genes. This is not seen in Arabidopsis, where most repressive marks are mutually exclusive, while in animals H3K27me3 can be found with DNA methylation [78], as well as H3K9me3 which acts as a binding site for heterochromatin protein 1 [79]. Like other organisms epigenetic regulation of genic features therefore provides diatoms with the potential to adapt to changing environments, likely shaping their genomes over short time scales. Conservation of the epigenetic machinery in diatoms is a unique opportunity to gain insights into the processes underpinning phenotypic plasticity and to explore their role in evolutionary processes.
Conclusions and future directions Diatoms are particularly interesting as model organisms for understanding the biology and evolution of eukaryotes because they combine many features believed previously to be unique to different lineages. With the advent of new sequencing technologies and lower costs, we have today a privileged access to the invisible planktonic life in the ocean within which diatoms play a wide range of key roles. Single cell sequencing is emerging as a powerful technology for learning more about such species in terms of their genome structure and function [80,81] and is certainly an important direction to take in the future considering the diversity and heterogeneity found in plankton communities. This is particularly important for those species that cannot be maintained under laboratory conditions, which represent the vast majority Another non negligible benefit from this technology is the potential possibility to analyze diatoms with interacting partners, such as symbionts, parasites, bacteria, and viruses [38,82] to better define community-level interactions and the role they play in food webs and biogeochemical cycles. Considering the vast extent of diatom diversity [37,83], it is important to work not only on elite experimental models but also to diversify towards less tractable species. This is one of the major objectives of an exciting initiative supported recently by the Gordon and Betty Moore Foundation that has gathered more than 150 laboratories to bring together their expertise to consolidate or establish new experimental model systems, in particular Current Opinion in Plant Biology 2017, 36:46–55
diatoms (Marine Microbial Initiative; URL: https:// www.moore.org/initiative-strategy-detail?initiativeId= marine-microbiology-initiative) [32]. Efforts also need to be made to refine analysis of gene expression data in light of recent studies indicating that diatoms may be natural hybrids [42,47]. For example, new bioinformatics tools will be needed to detect cases of mono-allelic gene expression or allele exclusion to fully understand their contribution to transcriptional regulation of genes and subsequent phenotypic variation, particularly in natural environments. The advent of technologies to assess the extent of genetic and epigenetic phenomena in driving diatom adaptations to changing environments over evolutionarily-relevant timescales will be equally critical for assessing the fate of diatoms in a future ocean modified by human-induced climate change.
Acknowledgements We acknowledge funding from the European Research Council, Louis D Foundation, Gordon and Betty Moore Foundation, Oceanomics to CB and the National Centre for Scientific Research to LT. AR was funded by a MEMO-LIFE International PhD Fellowship Program. We thank Atsuko Tanaka for the images presented in Figure 1.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.pbi.2017.02.001.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Lazarus D, Barron J, Renaudie J, Diver P, Turke A: Cenozoic planktonic marine diatom diversity and correlation to climate change. PLoS One 2014, 9:e84857.
2.
Kooistra WH, Medlin LK: Evolution of the diatoms (Bacillariophyta). IV. A reconstruction of their age from small subunit rRNA coding regions and the fossil record. Mol Phylogenet Evol 1996, 6:391-407.
3.
Keeling PJ: The endosymbiotic origin, diversification and fate of plastids. Philos Trans R Soc Lond B Biol Sci 2010, 365:729-748.
4.
Gould SB, Waller RF, McFadden GI: Plastid evolution. Annu Rev Plant Biol 2008, 59:491-517.
5.
Field CB, Behrenfeld MJ, Randerson JT, Falkowski P: Primary production of the biosphere: integrating terrestrial and oceanic components. Science 1998, 281:237-240.
6.
Patil S, Moeys S, von Dassow P, Huysman MJ, Mapleson D, De Veylder L, Sanges R, Vyverman W, Montresor M, Ferrante MI: Identification of the meiotic toolkit in diatoms and exploration of meiosis-specific SPO11 and RAD51 homologs in the sexual species Pseudo-nitzschia multistriata and Seminavis robusta. BMC Genom 2015, 16:930.
7.
Huysman MJ, Martens C, Vandepoele K, Gillard J, Rayko E, Heijde M, Bowler C, Inze D, Van de Peer Y, De Veylder L et al.: Genome-wide analysis of the diatom cell cycle unveils a novel type of cyclins involved in environmental signaling. Genome Biol 2010, 11:R17. www.sciencedirect.com
Diatom genomics and epigenomics Tirichine, Rastogi and Bowler 53
8.
Zofall M, Grewal SI: Swi6/HP1 recruits a JmjC domain protein to facilitate transcription of heterochromatic repeats. Mol Cell 2006, 22:681-692.
9.
Falkowski PG: Evolution of the nitrogen cycle and its influence on the biological sequestration of CO2 in the ocean. Nature 1997, 387:272-275.
10. Treguer PJ, De La Rocha CL: The world ocean silica cycle. Ann Rev Mar Sci 2013, 5:477-501. 11. Katz MEF, Finkel ZV, Grzebyk D, Knoll AH, Falkowski PG: Evolutionary trajectories and biogeochemical impacts of marine eukaryotic phytoplankton. Annu Rev Ecol Evol Syst 2004, 35:523-556. 12. Martin KR: The chemistry of silica and its potential health benefits. J Nutr Health Aging 2007, 11:94-97. 13. Wachter H, Lechleitner M, Artner-Dworzak E, Hausen A, Jarosch E, Widner B, Patsch J, Pfeiffer K, Fuchs D: Diatomaceous earth lowers blood cholesterol concentrations. Eur J Med Res 1998, 3:211-215. 14. Danil de Namor AF, El Gamouz A, Frangie S, Martinez V, Valiente L, Webb OA: Turning the volume down on heavy metals using tuned diatomite. A review of diatomite and modified diatomite for the extraction of heavy metals from water. J Hazard Mater 2012, 241–242:14-31. 15. Kroger N, Poulsen N: Diatoms-from cell wall biogenesis to nanotechnology. Annu Rev Genet 2008, 42:83-107. 16. Tanaka T, Maeda Y, Veluchamy A, Tanaka M, Abida H, Marechal E, Bowler C, Muto M, Sunaga Y, Tanaka M et al.: Oil accumulation by the oleaginous diatom Fistulifera solaris as revealed by the genome and transcriptome. Plant Cell 2015, 27:162-176. Sequencing of an oleaginous pennate diatom revealed an allodiploid genome structure with an unusual pattern of expression which might be due to the simultaneous growth and oil accumulation. 17. Raposo MF, de Morais RM, Bernardo de Morais AM: Bioactivity and applications of sulphated polysaccharides from marine microalgae. Mar Drugs 2013, 11:233-252. 18. Specht EA, Mayfield SP: Algae-based oral recombinant vaccines. Front Microbiol 2014, 5:60.
Evolution and metabolic significance of the urea cycle in photosynthetic diatoms. Nature 2011, 473:203-207. The authors report in diatoms genetic evidence for the significance of ornithine urea cycle which is absent from green algae and plants. 27. Montsant A, Jabbari K, Maheswari U, Bowler C: Comparative genomics of the pennate diatom Phaeodactylum tricornutum. Plant Physiol 2005, 137:500-513. 28. Rayko E, Maumus F, Maheswari U, Jabbari K, Bowler C: Transcription factor families inferred from genome sequences of photosynthetic stramenopiles. New Phytol 2016, 188:52-66. 29. Fortunato AE, Jaubert M, Enomoto G, Bouly JP, Raniello R, Thaler M, Malviya S, Bernardes JS, Rappaport F, Gentili B et al.: Diatom phytochromes reveal the existence of far-red-lightbased sensing in the ocean. Plant Cell 2016, 28:616-628. The authors demonstrated using a genetic approach the presence of red/ far red light sensing in diatoms mediated by phytochromes. 30. Maumus F, Allen AE, Mhiri C, Hu H, Jabbari K, Vardi A, Grandbastien MA, Bowler C: Potential impact of stress activated retrotransposons on genome evolution in a marine diatom. BMC Genom 2009, 10:624. 31. Galachyants YP, Zakharova YR, Petrova DP, Morozov AA, Sidorov IA, Marchenkov AM, Logacheva MD, Markelov ML, Khabudaev KV, Likhoshway YV et al.: Sequencing of the complete genome of an araphid pennate diatom Synedra acus subsp. radians from Lake Baikal. Dokl Biochem Biophys 2015, 461:84-88. 32. Keeling PJ, Burki F, Wilcox HM, Allam B, Allen EE, Amaral Zettler LA, Armbrust EV, Archibald JM, Bharti AK, Bell CJ et al.: The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing. PLoS Biol 2014, 12:e1001889. A community effort which led to the creation of a single database with more than 650 assembled and functionally annotated genomes from a range of protists. 33. Karsenti E, Acinas SG, Bork P, Bowler C, De Vargas C, Raes J, Sullivan M, Arendt D, Benzoni F, Claverie JM et al.: A holistic approach to marine eco-systems biology. PLoS Biol 2011, 9:e1001177.
19. Nazmi A, Hauck R, Davis A, Hildebrand M, Corbeil LB, Gallardo RA: Diatoms and diatomaceous earth as novel poultry vaccine adjuvants Poult Sci.
34. Allen AE, Laroche J, Maheswari U, Lommer M, Schauer N, Lopez PJ, Finazzi G, Fernie AR, Bowler C: Whole-cell response of the pennate diatom Phaeodactylum tricornutum to iron starvation. Proc Natl Acad Sci U S A 2008, 105:10438-10443.
20. Bowler C, Allen AE, Badger JH, Grimwood J, Jabbari K, Kuo A, Maheswari U, Martens C, Maumus F, Otillar RP et al.: The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 2008, 456:239-244. Genome sequencing of the first pennate diatom revealed an important rate of gene diversification compared to yeast and metazoans as well as expansion of gene families and presence of genes of bacterial origin.
35. Morrissey J, Sutak R, Paz-Yepes J, Tanaka A, Moustafa A, Veluchamy A, Thomas Y, Botebol H, Bouget FY, McQuaid JB et al.: A novel protein, ubiquitous in marine phytoplankton, concentrates iron at the cell surface and facilitates uptake. Curr Biol 2015, 25:364-371.
21. Moustafa A, Beszteri B, Maier UG, Bowler C, Valentin K, Bhattacharya D: Genomic footprints of a cryptic plastid endosymbiosis in diatoms. Science 2009, 324:1724-1726. Evidence for green genes in diatoms. 22. Armbrust EV, Berges JA, Bowler C, Green BR, Martinez D, Putnam NH, Zhou S, Allen AE, Apt KE, Bechner M et al.: The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 2004, 306:79-86. This paper describes genome structure and features of the first centric diatom to be sequenced. 23. Liu H, Fu Y, Li B, Yu X, Xie J, Cheng J, Ghabrial SA, Li G, Yi X, Jiang D: Widespread horizontal gene transfer from circular single-stranded DNA viruses to eukaryotic genomes. BMC Evol Biol 2011, 11:276. 24. Armbrust EV: The life of diatoms in the world’s oceans. Nature 2009, 459:185-192. 25. Bowler C, Vardi A, Allen AE: Oceanographic and biogeochemical insights from diatom genomes. Ann Rev Mar Sci 2010, 2:333-365. 26. Allen AE, Dupont CL, Obornik M, Horak A, Nunes-Nesi A, McCrow JP, Zheng H, Johnson DA, Hu H, Fernie AR et al.: www.sciencedirect.com
36. Montsant AA, Coesel AE, De Martino S, Falciatore A, Mangogna A, Siaut M, Heijde M, Jabbari M, Maheswari K, Rayko U et al.: Identification and comparative genomic analysis of signaling and regulatory components in the diatom Thalassiosira pseudonana. J Phycol 2007, 43:585-604. 37. Malviya S, Scalco E, Audic S, Vincent F, Veluchamy A, Poulain J, Wincker P, Iudicone D, de Vargas C, Bittner L et al.: Insights into global diatom distribution and diversity in the world’s ocean. Proc Natl Acad Sci U S A 2016:E1516-E1525. 38. Lima-Mendez G, Faust K, Henry N, Decelle J, Colin S, Carcillo F, Chaffron S, Ignacio-Espinosa JC, Roux S, Vincent F et al.: Ocean plankton. Determinants of community structure in the global plankton interactome. Science 2015, 348:1262073. 39. Finlay BJ: Global dispersal of free-living microbial eukaryote species. Science 2002, 296:1061-1063. 40. d’Ovidio F, De Monte S, Alvain S, Dandonneau Y, Levy M: Fluid dynamical niches of phytoplankton types. Proc Natl Acad Sci U S A 2010, 107:18366-18370. 41. Sjoqvist C, Godhe A, Jonsson PR, Sundqvist L, Kremp A: Local adaptation and oceanographic connectivity patterns explain genetic differentiation of a marine diatom across the North Sea-Baltic Sea salinity gradient. Mol Ecol 2015, 24:2871-2885. Current Opinion in Plant Biology 2017, 36:46–55
54 Genome studies and molecular genetics
42. Rynearson TA, Armbrust EV: Maintenance of clonal diversity during a spring bloom of the centric diatom Ditylum brightwellii. Mol Ecol 2005, 14:1631-1640. Molecular evidence for hybridization in diatoms. 43. Casteleyn G, Leliaert F, Backeljau T, Debeer AE, Kotaki Y, Rhodes L, Lundholm N, Sabbe K, Vyverman W: Limits to gene flow in a cosmopolitan marine planktonic diatom. Proc Natl Acad Sci U S A 2010, 107:12952-12957. 44. Chen GR: Genetically distinct populations of a diatom co-exist during the North Atlantic spring bloom. Limnol Oceanogr 2016, 61(6):2165-2179 http://dx.doi.org/10.1002/lno.10361. 45. Casteleyn G, Adams NG, Vanormelingen P, Debeer AE, Sabbe K, Vyverman W: cast(Bacillariophyceae): genetic and morphological evidence. Protist 2009, 160:343-354. 46. D’Alelio D, Amato A, Kooistra WH, Procaccini G, Casotti R, Montresor M: Internal transcribed spacer polymorphism in Pseudo-nitzschia multistriata (Bacillariophyceae) in the Gulf of Naples: recent divergence or intraspecific hybridization? Protist 2009, 160:9-20. 47. Casteleyn G, Adams NG, Vanormelingen P, Debeer AE, Sabbe K, Vyverman W: Natural hybrids in the marine diatom Pseudonitzschia pungens (Bacillariophyceae): genetic and morphological evidence. Protist 2009, 160:343-354. 48. Hart MC, Green DH, Bresnan E, Bolch CJ: Large subunit ribosomal RNA gene variation and sequence heterogeneity of Dinophysis (Dinophyceae) species from Scottish coastal waters. Harmful Algae 2007, 6:271-287. 49. Edvardsen B, Shalchian-Tabrizi K, Jakobsen KS, Medlin LK, Dahl E, Brubak S, Paasche E: Genetic variability and molecular phylogeny of Dinophysis species (Dinophyceae) from Norwegian waters inferred from single cell analyses of rDNA. J Phycol 2003, 39:395-408. 50. Keverne B: Monoallelic gene expression and mammalian evolution. Bioessays 2009, 31:1318-1326. 51. Shykind BM: Regulation of odorant receptors: one allele at a time. Hum Mol Genet 2005, 14 Spec No 1:R33-R39. 52. De Riso V, Raniello R, Maumus F, Rogato A, Bowler C, Falciatore A: Gene silencing in the marine diatom Phaeodactylum tricornutum. Nucleic Acids Res 2009, 37:e96. 53. Daboussi F, Leduc S, Marechal A, Dubois G, Guyot V, PerezMichaut C, Amato A, Falciatore A, Juillerat A, Beurdeley M et al.: Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology. Nat Commun 2014, 5:3831. 54. Hamilton ML, Haslam RP, Napier JA, Sayanova O: Metabolic engineering of Phaeodactylum tricornutum for the enhanced accumulation of omega-3 long chain polyunsaturated fatty acids. Metab Eng 2014, 22:3-9. 55. Niu YF, Wang X, Hu DX, Balamurugan S, Li DW, Yang WD, Liu JS, Li HY: Molecular characterization of a glycerol-3-phosphate acyltransferase reveals key features essential for triacylglycerol production in Phaeodactylum tricornutum. Biotechnol Biofuels 2016, 9:60. 56. Falciatore A, Casotti R, Leblanc C, Abrescia C, Bowler C: Transformation of nonselectable reporter genes in marine diatoms. Mar Biotechnol (N Y) 1999, 1:239-251. 57. Karas BJ, Diner RE, Lefebvre SC, McQuaid J, Phillips AP, Noddings CM, Brunson JK, Valas RE, Deerinck TJ, Jablanovic J et al.: Designer diatom episomes delivered by bacterial conjugation. Nat Commun 2015, 6:6925. The authors report a conjugation-based protocol for P. tricornutum transformation based on transfer of plamsids from Escherichia coli to diatoms. 58. Niu YF, Yang ZK, Zhang MH, Zhu CC, Yang WD, Liu JS, Li HY: Transformation of diatom Phaeodactylum tricornutum by electroporation and establishment of inducible selection marker. Biotechniques 2012, 52. 59. Miyahara M, Aoi M, Inoue-Kashino N, Kashino Y, Ifuku K: Highly efficient transformation of the diatom Phaeodactylum Current Opinion in Plant Biology 2017, 36:46–55
tricornutum by multi-pulse electroporation. Biosci Biotechnol Biochem 2013, 77:874-876. 60. Kaur S, Spillane C: Reduction in carotenoid levels in the marine diatom Phaeodactylum tricornutum by artificial microRNAs targeted against the endogenous phytoene synthase gene. Mar Biotechnol (N Y) 2015, 17:1-7. 61. Nymark M, Sharma AK, Sparstad T, Bones AM, Winge P: A CRISPR/Cas9 system adapted for gene editing in marine algae. Sci Rep 2016, 6:24951. 62. Maheswari U, Jabbari K, Petit JL, Porcel BM, Allen AE, Cadoret JP, De Martino A, Heijde M, Kaas R, La Roche J et al.: Digital expression profiling of novel diatom transcripts provides insight into their biological functions. Genome Biol 2010, 11: R85. 63. Bailleul B, Berne N, Murik O, Petroutsos D, Prihoda J, Tanaka A, Villanova V, Bligny R, Flori S, Falconet D et al.: Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms. Nature 2015, 524:366-369. Evidence for energetic exchanges between the plastid and mitochondria necessary for carbon fixation and growth in diatoms. 64. Huysman MJ, Fortunato AE, Matthijs M, Costa BS, Vanderhaeghen R, Van den Daele H, Sachse M, Inze D, Bowler C, Kroth PG et al.: AUREOCHROME1a-mediated induction of the diatom-specific cyclin dsCYC2 controls the onset of cell division in diatoms (Phaeodactylum tricornutum). Plant Cell 2013, 25:215-228. 65. Maumus F, Rabinowicz P, Bowler C, Rivarola M: Stemming epigenetics in marine stramenopiles. Curr Genom 2011, 12:357-370. 66. Rastogi A, Lin X, Lombard B, Loew D, Tirichine L: Probing the evolutionary history of epigenetic mechanisms: what can we learn from marine diatoms. AIMS Genet 2015, 2:173-191. 67. Veluchamy A, Lin X, Maumus F, Rivarola M, Bhavsar J, Creasy T, O’Brien K, Sengamalay NA, Tallon LJ, Smith AD et al.: Insights into the role of DNA methylation in diatoms by genome-wide profiling in Phaeodactylum tricornutum. Nat Commun 2013, 4. 68. Huff JT, Zilberman D: Dnmt1-independent CG methylation contributes to nucleosome positioning in diverse eukaryotes. Cell 2014, 156:1286-1297. The authors found a novel DNA methylatransferase that fulfils the function of animal dnmt1 methylating DNA in a GC context with clustered methylation over nucleosome linkers, contributing to nucleosome positionning between clusters. 69. Rastogi A, Murik O, Bowler C, Tirichine L: PhytoCRISP-Ex: a web-based and stand-alone application to find specific target sequences for CRISPR/CAS editing. BMC Bioinf 2016, 17:261. The authors built a user friendly software to search for target sequences in 13 phytoplankton species. 70. Traller JC, Cokus SJ, Lopez DA, Gaidarenko O, Smith SR, McCrow JP, Gallaher SD, Podell S, Thompson M, Cook O et al.: Genome and methylome of the oleaginous diatom Cyclotella cryptica reveal genetic flexibility toward a high lipid phenotype. Biotechnol Biofuels 2016, 9:258. 71. Rogato A, Richard H, Sarazin A, Voss B, Cheminant Navarro S, Champeimont R, Navarro L, Carbone A, Hess WR, Falciatore A: The diversity of small non-coding RNAs in the diatom Phaeodactylum tricornutum. BMC Genom 2014, 15:698. 72. Huang A, He L, Wang G: Identification and characterization of microRNAs from Phaeodactylum tricornutum by highthroughput sequencing and bioinformatics analysis. BMC Genom 2011, 12:337. 73. Norden-Krichmar TM, Allen AE, Gaasterland T, Hildebrand M: Characterization of the small RNA transcriptome of the diatom, Thalassiosira pseudonana. PLoS One 2011, 6:e22870. 74. Lopez-Gomollon S, Beckers M, Rathjen T, Moxon S, Maumus F, Mohorianu I, Moulton V, Dalmay T, Mock T: Global discovery and characterization of small non-coding RNAs in marine microalgae. BMC Genom 2014, 15:697. www.sciencedirect.com
Diatom genomics and epigenomics Tirichine, Rastogi and Bowler 55
75. Cruz de Carvalho MH, Sun HX, Bowler C, Chua NH: Noncoding and coding transcriptome responses of a marine diatom to phosphate fluctuations. New Phytol 2016, 210:497-510.
81. Roy RS, Price DC, Schliep A, Cai G, Korobeynikov A, Yoon HS, Yang EC, Bhattacharya D: Single cell genome analysis of an uncultured heterotrophic stramenopile. Sci Rep 2014, 4:4780.
76. Lin X, Tirichine L, Bowler C: Protocol: chromatin immunoprecipitation (ChIP) methodology to investigate histone modifications in two model diatom species. Plant Methods 2012, 8:48.
82. Riemann L, Steward GF, Azam F: Dynamics of bacterial community composition and activity during a mesocosm diatom bloom. Appl Environ Microbiol 2000, 66:578-587.
77. Veluchamy A, Rastogi A, Lin X, Lombard B, Murik O, Thomas Y, Dingli F, Rivarola M, Ott S, Liu X et al.: An integrative analysis of post-translational histone modifications in the marine diatom Phaeodactylum tricornutum. Genome Biol 2015, 16:102. The authors decribe for the first time the identification of several PTMs in a diatom and draw a chromatin landscape demonstrating the presence of a histone code sharing features with animals and plants and others unique to diatoms. 78. Vire E, Brenner C, Deplus R, Blanchon L, Fraga M, Didelot C, Morey L, Van Eynde A, Bernard D, Vanderwinden JM et al.: The Polycomb group protein EZH2 directly controls DNA methylation. Nature 2006, 439:871-874. 79. Ringrose L, Ehret H, Paro R: Distinct contributions of histone H3 lysine 9 and 27 methylation to locus-specific stability of polycomb complexes. Mol Cell 2004, 16:641-653. 80. Bhattacharya D, Price DC, Yoon HS, Yang EC, Poulton NJ, Andersen RA, Das SP: Single cell genome analysis supports a link between phagotrophy and primary plastid endosymbiosis. Sci Rep 2012, 2:356.
www.sciencedirect.com
83. Leblanc K, Arı´stegui J, Armand J, Assmy P, Beker B, Bode B, Breton E, Cornet V, Gibson J, Gosselin MP et al.: A global diatom database—abundance, biovolume and biomass in the world ocean. Earth Syst Sci Data 2012, 4:149-165. 84. Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987, 4:406-425. 85. Guillou L, Bachar D, Audic S, Bass D, Berney C, Bittner L, Boutte C, Burgaud G, de Vargas C, Decelle J et al.: The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote small subunit rRNA sequences with curated taxonomy. Nucleic Acids Res 2013, 41:D597-604. 86. Felsenstein J: Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985, 39:783-791. 87. Tamura K, Nei M, Kumar S: Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci U S A 2004, 101:11030-11035. 88. Kumar S, Stecher G, Tamura K: MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 2016, 33:1870-1874.
Current Opinion in Plant Biology 2017, 36:46–55
ScienceDirect Recent progress in diatom genomics and epigenomics Leila Tirichine, Achal Rastogi and Chris Bowler 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.
Address Ecole Normale Supe´rieure, PSL Research University, Institut de Biologie de l’Ecole Normale Supe´rieure (IBENS), CNRS UMR 8197, INSERM U1024, 46 rue d’Ulm, F-75005 Paris, France Corresponding author: Tirichine, Leila ([email protected])
Current Opinion in Plant Biology 2017, 36:46–55 This review comes from a themed issue on Genome studies and molecular genetics Edited by Ian Henderson and Korbinian Schneeberger
http://dx.doi.org/10.1016/j.pbi.2017.02.001 1369-5266/ã 2017 Elsevier Ltd. All rights reserved.
Introduction Diatoms are highly diverse and cosmopolitan single celled eukaryotes of the Stramenopile lineage found in nearly all aquatic habitats including fresh and marine waters and moist soil. Diatoms date back to at least the early Jurassic (185 Ma ago), they survived the endCretaceous mass extinction relatively unscathed, and underwent a major diversification during the Cenozoic [1] that led to them becoming one of the major groups of phytoplankton in contemporary oceans [2]. They are believed to be derived from a serial secondary endosymbiosis approximately 1 billion years ago [3,4] in which a heterotrophic eukaryotic host domesticated photosynthetic eukaryotic cells. Current Opinion in Plant Biology 2017, 36:46–55
Like higher plants, diatoms are obligately photosynthetic. Current estimates indicate that they contribute around 40% of primary production in the ocean and 20% of global carbon fixation [5]. They are therefore a major component providing organic carbon to higher trophic levels in marine food webs, as well as participating in the global carbon cycle. Another feature of diatoms is their elaborate cell wall known as the frustule, composed of amorphous silica, which influences multiple critical aspects of diatom life histories, including cell division [6,7], susceptibility to grazing [8], and sinking rates from sunlit upper layers to the ocean interior. The latter process contributes to carbon export and is an extremely important biological process that removes carbon from the global carbon cycle over geological time scales [9], ultimately contributing to the generation of oil and gas fossil fuel reserves. Furthermore, the mineralization of silicon by diatoms is a major component of the silica biogeochemical cycle [10]. Fossilized frustules are valuable paleo tracers for geologists [11], and have accumulated in vast deposits of diatomaceous earth that are quarried for a range of applications [12–14]. Diatom frustules show a wide diversity of morphologies (Figure 1), which have been the basis of traditional diatom taxonomy, dividing them into two orders: centric diatoms that have radial symmetry and pennate diatoms that are elongate with thin ellipses. Centric diatoms are further divided into polar centrics (Mediophyceae) and radial centrics (Coscinodiscophyceae), and pennate diatoms into raphid (Bacillariophyceae) and araphid diatoms (Fragilariophyceae) which is based on the presence or absence of a raphe in the valve, which facilitates movement. Diatoms are also of interest for several applications including the production of therapeutics, biofuels and biopolymers [15,16]. They are increasingly being used as molecular farms to produce several molecules such as polyunsaturated fatty acids, carotenoids, polysaccharides, vitamins, and sterols [17], as well as vaccines and their adjuvants [18,19]. Today, materials from diatoms provide numerous items ranging from toothpaste, nail polish, filters for swimming pools and fountains, building materials, biofuel and diatomaceous earth for organic pest control. The ecological, evolutionary and biotechnological interests of diatoms have attracted the attention of the scientific community, leading to a significant increase in published articles that have led to tangible advances in knowledge, particularly enabled by genomics. Here we www.sciencedirect.com
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Figure 1
The morphological diversity of diatoms revealed by different microscopy techniques. Chain of pennate diatoms (top left) and individual centric diatom (top right) observed by epifluorescence microscopy. Cell impermeable Alexa Fluor 546 conjugates reveal extracellular structures (green), red chlorophyll autofluorescence reveals chloroplasts, and DAPI stains the nucleus in blue (only in top left panel). The image in top right is of Planktoniella, an open ocean genus characterized by an elaborate spoke-like extracellular structure that may facilitate buoyancy or provide protection from grazers. The lower two panels show scanning electron micrographs of the nanostructured silicified frustules of a pennate diatom (Fragilariopsis doliolus) (bottom left) and a centric diatom (possibly Thalassiosira) (bottom right). The diatoms were collected during the Tara Oceans expedition and images were made by Atsuko Tanaka. Scale bars are indicated.
present an overview of recent progress in diatom genomics, comparative genomics and epigenomics. We particularly review the different components of chromatinlevel regulation and highlight examples showing their contribution to genome regulation. Such examples are placed in the context of a chimeric genome derived from the unusual evolutionary history of diatoms that has brought together genes of red and green algal origin as well as from bacteria [20,21,22]. Allele specific expression is an additional feature that may permit further phenotypic plasticity and thus help diatoms to thrive in dynamic highly unstable environments.
Tiny but complex Thalassiosira pseudonana and Phaeodactylum tricornutum were the first diatom genomes to be sequenced, www.sciencedirect.com
representing centric and pennate diatoms, respectively [20,22]. Both species are on the small side for diatoms (less than 10 mm), as are their genomes, on the order of 30 Mb. Nonetheless, they each encode a plethora of genes, around 12 000, encoding diverse and complex functions likely acquired through their evolutionary history combining genes from exosymbiont as well as from algal endosymbionts and bacteria [20,21,22,23]. The unusual gene repertoire of diatoms has been proposed to underpin their ecological success in the contemporary ocean [24,25]. For example, diatom genomes have genes encoding a metazoan-like ornithine urea cycle that is absent from plants [20,26,27], large numbers of diatom-specific cyclins, heat shock transcription factors [7,28], and far-red light sensors related to phytochromes [29]. Current Opinion in Plant Biology 2017, 36:46–55
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Diatom genomes were furthermore shown to be compact with small gene sizes and intergenic regions, and a significant proportion of transposable elements (TEs) which predominantly belong to class I TEs, in particular the LTR retrotransposon superfamily [30]. Examination of LTR retro-elements of the copia type in both P. tricornutum and T. pseudonana revealed the existence of seven groups of diatom-specific TEs named CoDi (Copia-like elements from diatoms) [30]. Some of the CoDi groups were shown to be expressed under specific conditions in P. tricornutum and T. pseudonana suggesting a role of TEs in adaptation and diversification of diatoms [25,30]. Although challenging because of their repetitive sequences and dynamic behavior within a genome, TE annotation should be further pursued in diatoms to better understand their influence in diatom evolution, speciation, and adaptation to the environment.
Diatom eclectic diversity More than ten diatom whole genome sequences including the first freshwater diatom [31] are today available or being generated (Table 1). These genome sequences have been further enriched with transcriptomes from the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) [32] which has put together over 650 assembled, functionally annotated transcriptomes amongst which 92 diatom species (Figure 2, Table S1). From the most abundant diatom genera in the Tara Oceans global ocean plankton sampling survey [33], seven additional diatom transcriptomes have been generated by Genoscope (from Shionodiscus, Synedra, Pleurosigma, Navicula, Haslea, Guinardia and Minidiscus) (Table 1). This growing database of diatom sequences represents a valuable resource for functional and comparative genomics studies that can help understand
evolutionary histories and reveal the metabolic peculiarities that distinguish them from other species [26,34–36]. Genomics studies have so far examined only a tiny portion of the existing diversity of diatoms. A metabarcoding approach based on 18s rDNA sequences obtained from environmental samples collected from major oceanic provinces during the Tara Oceans expedition revealed 63 371 unique diatom-derived barcodes, potentially representing up to 4748 taxa [37], including a considerable number of abundant yet unidentifiable species. An important objective in future years will be to bring representatives of this unexplored diatom diversity into culture. The abundance of prokaryotes, eukaryotes and viruses at multiple sampling sites was further used to generate an ocean plankton interactome that revealed that collaboration rather than competition between organisms was more prevalent [38]. A conspicuous exception were the diatoms, that appear to exclude other organisms and thus to be more competitive than other phytoplankton groups [38]. Because diatoms (like other marine microbial microorganisms) are constituted by large populations with vast possibilities for geographic dispersal, rapid growth rates, and mainly asexual reproduction, they are classically considered as having a continuous distribution and weak biogeographic population structures [39]. However, the oceans are not homogeneous because of seascape topography and ocean circulation acting over multiple scales both horizontally and vertically, resulting in localized micro-environments that shape the diversity of the biota within them and create barriers to dispersal [40]. Indeed, changing conditions over time and space can lead to the appearance of specific endemic taxa that are able to
Table 1 Genomic features of the first sequenced diatom genomes. One important finding distinguishing pennate from centric diatoms is the fraction of genes (40%) that are not shared between the two lineages which can lead to differences such as the preference in codon usage in P. tricornutum that is different from T. pseudonana [36]. P. tricornutum seems to possess the smallest genome size as well as number of genes, the largest being the genome of Pseudo-nitzschia multiseries (Table 1). Some of the earliest sequenced genomes such as T. pseudonana and P. tricornutum have seen their annotation improved and several reiterations have been made since the first annotated draft. Phaeodactylum tricornutum annotation 3 (Phatr3) available at Ensembl revealed over 1000 new genes and transposable elements and improved significantly existing gene models (Rastogi et al., in review) Genome feature
Nuclear genome size (MB) Number of chromosome(s)/scaffold(s) Number of genes Average length of a gene (bp) Average length of an exon (bp) Average length of an intron (bp) Average protein length (aa) Average exon density per gene Current annotation version
Centric diatoms
Pennate diatoms
Thalassiosira pseudonana
Thalassiosira oceanica
Phaeodactylum tricornutum
Fragilariopsis cylindrus
Pseudo-nitzschia multiseries
32.1 65 11 776 1674 571 129 464 2.6 v3 (2007)
92.0 51 656 34 500 1256 464 146 354 2.3 v1 (2014)
27.4 88 12 177 1624 886 142 495 1.7 v3 (2016)
80.5 271 27 137 1572 615 256 409 2.1 v1 (2008)
218.73 4890 19 703 1522 509 229 377 2.4 v1 (2011)
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Figure 2
The evolutionary divergence of diatoms. Sequences from 57 diatom species, whose transcriptome sequences are available in the MMETSP database and 18S gene reference is available in PR2 database, were used to generate a phylogenetic tree using the Neighbor-Joining method [84]. The tree shows the phylogenetic association between the newly sequenced diatom species with model species such as P. tricornutum and T. pseudonana. The tree was generated using 18S rRNA gene sequences taken from the PR2 database [85]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [86]. The evolutionary distances were computed using the Maximum Composite Likelihood method [87] and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA7 [88]. Boxed in red color, the tree is rooted to Bolidophytes, sister lineage to diatoms. Node prefixed by black diamonds indicates pennate diatom species and others are centric diatoms. The tree is clustered into four subclasses of diatom, which is based on the valve organization of the diatom cell.
outcompete conspecifics and evolve into distinct species [41,42,43]. Furthermore, different diatom species can coexist within the same water column spanning a range of depths during a bloom suggesting that the ocean contains a pool of standing stock genetic diversity upon which selection can act [44]. Diatoms are known to display high diversification rates which have been proposed to be generated at least in part as a result of occasional sexual reproduction events between genetically distinguishable taxa, thus creating natural hybrids that can disperse diversity within a population [45]. The genome sequences of both Fistulifera solaris and Fragilariopsis cylindrus have provided further molecular evidence for such a phenomenon [16] (Mock et al., in press). For www.sciencedirect.com
example, in F. cylindrus 45% of divergent alleles showed an allele specific expression in at least one of the tested conditions, suggesting the importance of differential allele expression in fine tuning gene regulation in response to the environment (Mock et al., in press). It therefore seems possible that heterosis may be pervasive in natural diatom populations. Natural hybridization has been proposed previously to play a role in generating diversity within multiple marine micro-eukaryotes, including diatoms [16,46,47] and dinoflagellates [48,49], suggesting its potential role in creating heterogeneity and thus diversity within a species and/or genus. Bi- and mono-allelism, defined as the expression of either both alleles or only one allele in a diploid cell, has been Current Opinion in Plant Biology 2017, 36:46–55
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shown to occur in specific classes of genes in many organisms, expanding their diversity and adaptability, playing thus a significant role in their evolution [50]. This is typically the case for genes encoding olfactory and immune receptors in mammals, allowing progeny recognition and survival, and increasing disease resistance [51]. It will be of great interest to examine the extent of the phenomenon in diatoms, both in laboratory and natural conditions.
A growing toolbox to determine gene function in diatoms Sequencing diatom genomes and transcriptomes is one step towards decoding and understanding their genome, physiology, ecology, and evolutionary history. But another challenge is to determine the function of individual genes, in particular those that have no functional information assigned to them following in silico annotation. In a case study, the fact that a significant proportion of P. tricornutum genes (13%) are diatom-specific [20] (Rastogi et al., in revision) means that, in the overwhelming majority of cases, we have no functional information about the proteins encoded by them. With this in mind, over the last two decades the diatom community has developed tools and methods to explore gene function. Both P. tricornutum and T. pseudonana are now amenable to reverse genetics [52,53], although the superior tools developed for P. tricornutum have made it the experimental system of choice for most studies. Interest in using P. tricornutum as a cell factory for the production of highvalue products is facilitating this progress [54,55], as is the use of the species to explore diatom biology in an ecological context. Although found only in coastal areas and considered to be of limited ecological relevance, P. tricornutum has nonetheless demonstrated its usefulness in this regards because of the conservation of basic mechanisms among diatoms. This is illustrated by a study of iron uptake pathways in P. tricornutum which were shown to be similar to other low iron quota ecologically relevant diatoms, including pennate and centric species [35]. P. tricornutum is a robust laboratory species that can grow axenically in a range of culture media. It is less silicified compared to other diatoms, which is a considerable advantage for introducing exogenous DNA; in fact the species is amenable for transformation by microparticle gun bombardment [56], conjugation with Escherichia coli, and electroporation [57,58,59]. Different knock-down/ out strategies are feasible in P. tricornutum including RNA interference, TALEN, Artificial MicroRNA, and lately CRISPR cas9 editing [52,53,60,61]. Furthermore, digital gene expression approaches have been designed that permit the identification of candidate genes of potential importance for responding to a range of environmentallyrelevant signals [62]. Such examples include studies using RNAi silencing to characterize the peculiar urea cycle found in diatoms [26], the iron starvation induced Current Opinion in Plant Biology 2017, 36:46–55
protein ISIP2a, a protein that was shown to concentrate Fe(III) at the cell surface facilitating its uptake in iron limiting conditions [35], an alternative oxidase involved in extensive cross-talk between mitochondria and chloroplasts that improves photosynthetic efficiency [63], and diatom-specific cyclins implicated in the control of cell division by light [7,35,63,64]. More recently, several genes have been functionally characterized using knockout tools, including the far red light sensing DPH protein in P. tricornutum and T. pseudonana [29] which is surprising because of the absence or scarcity of such long wavelengths of light in subsurface waters. Altogether, these different studies indicate that the investigated genes contribute to the ecological success of diatoms in the ocean.
Conservation and surprises in the epigenetic machinery of diatoms In silico analyses of available diatom whole genome sequences have revealed the existence of nearly all components of the epigenetic machinery normally found in higher eukaryotes [65,66], suggesting the ancient origin of this mode of genome regulation. A striking example is cytosine methylation, which was first detected by HPLC and then molecularly using McrBC digestion coupled to microarray hybridization, and bisulfite sequencing [67]. The study by Veluchamy et al. found that in P. tricornutum a low percentage of DNA methylation can be found over around 5% of genes, intergenic regions, and TEs, and in all contexts (CG, CHH and CHG, where H can be any nucleotide except G) suggesting that this is not a plantspecific feature, as previously thought [67], but in fact evolved long before in eukaryote evolution. Later work found similar patterns of DNA methylation in both T. pseudonana, F. cylindrus and more recently in Cyclotella cryptica where 61% of the genome was found methylated, which is the highest methylation level currently known in a diatom species [68,69,70]. Furthermore, a dynamic regulation of DNA methylation was observed in response to nitrate starvation, with both genes and TEs being differentially methylated, suggesting a role in surviving nutrient limitation in the ocean [67]. Some DNA methylated regions (DMRs) are highly packed with small RNAs [71] suggesting an RNA-directed DNA methylation process which is further supported by in silico evidence of genes encoding DNA polymerases PolIV and V in P. tricornutum (Tirichine, unpublished). Non-coding RNAs have in fact been reported in diatoms by several groups although an in-depth functional characterization is still lacking [71–75]. Canonical miRNAs which have a typical complex for generating precursor miRNAs (Drosha) were not detected in the studied diatoms, but only 25–30 nt-long RNAs found to map principally to repeats, TEs and a subset of genes. Interestingly, some of these small RNAs display a 180 nt-long periodic distribution at several locations in the genome of P. tricornutum [71] suggesting a possible nucleosome-mediated www.sciencedirect.com
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distribution of sRNAs as described previously for DNA methylation [68]. The latter study showed a clustered methylation over nucleosome linkers contributing therefore to nucleosome positioning [68] between clusters which might also be mediated by small RNAs. This periodic methylation was identified in multiple species of Mamiellophyceae which might be the result of their small cell size and compact genome, features shared with diatoms, likely facilitating nuclear functions in extremely high DNA densities [68].
Furthermore, post translational modifications (PTMs) of histones were detected using mass spectrometry in both P. tricornutum and T. pseudonana [76,77]. An interesting finding is that P. tricornutum combines PTMs specific to plants or animals which might reflect the chimeric nature of its genome [77]. Whole genome mapping of a few key PTMs in P. tricornutum revealed a conserved histone code more similar to animals than plants [77] (Figure 3). This histone or epigenetic code implies that histone marks and DNA methylation co-occur to determine chromatin states
Figure 3
Chromatin features in P. tricornutum. Two snapshots of a 12 kb region (a) and 3 kb region (b) from P. tricornutum genome showing the distribution of five chromatin marks (tracks H3K4me2, H3K9/14Ac, H3K9me2, H3K9me3, and H3K27me3), nucleosome positioning (track H3; using anti-H3 antibody [77]), as well as DNA methylation (track DNA meth.) [67,68] over genes and transposable elements (TEs). These snapshots show the presence of a histone code that regulates gene expression, with genes marked by active marks while TEs are co-marked by repressive marks known to be mutually exclusive in other lineages such as plants. Repressive marks such as H3K27me3, H3K9me2/3 and DNA methylation co-localize on TEs and sometimes on genes (b) which lead to the repression of their expression (indicated by reduced RPKM values) [77]. Note spreading of H3K9me2 to the neighboring gene which causes a diminution in its expression. The active marks H3K4me2 and H3K9/ 14Ac co-localize on genes that are actively transcribed. www.sciencedirect.com
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that are either repressive or active, leading to expression or repression of genes and transposable elements. One striking pattern is that of H3K27me3, a mark deposited by polycomb protein complex 2 (PRC2) which targets mainly TEs and only 10% of genes [77]. This is different from plants such as Arabidopsis where the mark covers only genes but is similar to animals and the filamentous fungus Neurospora crassa, suggesting similar roles in defining a facultative heterochromatin [77]. The same study revealed another interesting feature which is the cooccurrence of H3K27me3 with two other repressive marks (H3K9me2/me3), as well as DNA methylation over TEs and genes. This is not seen in Arabidopsis, where most repressive marks are mutually exclusive, while in animals H3K27me3 can be found with DNA methylation [78], as well as H3K9me3 which acts as a binding site for heterochromatin protein 1 [79]. Like other organisms epigenetic regulation of genic features therefore provides diatoms with the potential to adapt to changing environments, likely shaping their genomes over short time scales. Conservation of the epigenetic machinery in diatoms is a unique opportunity to gain insights into the processes underpinning phenotypic plasticity and to explore their role in evolutionary processes.
Conclusions and future directions Diatoms are particularly interesting as model organisms for understanding the biology and evolution of eukaryotes because they combine many features believed previously to be unique to different lineages. With the advent of new sequencing technologies and lower costs, we have today a privileged access to the invisible planktonic life in the ocean within which diatoms play a wide range of key roles. Single cell sequencing is emerging as a powerful technology for learning more about such species in terms of their genome structure and function [80,81] and is certainly an important direction to take in the future considering the diversity and heterogeneity found in plankton communities. This is particularly important for those species that cannot be maintained under laboratory conditions, which represent the vast majority Another non negligible benefit from this technology is the potential possibility to analyze diatoms with interacting partners, such as symbionts, parasites, bacteria, and viruses [38,82] to better define community-level interactions and the role they play in food webs and biogeochemical cycles. Considering the vast extent of diatom diversity [37,83], it is important to work not only on elite experimental models but also to diversify towards less tractable species. This is one of the major objectives of an exciting initiative supported recently by the Gordon and Betty Moore Foundation that has gathered more than 150 laboratories to bring together their expertise to consolidate or establish new experimental model systems, in particular Current Opinion in Plant Biology 2017, 36:46–55
diatoms (Marine Microbial Initiative; URL: https:// www.moore.org/initiative-strategy-detail?initiativeId= marine-microbiology-initiative) [32]. Efforts also need to be made to refine analysis of gene expression data in light of recent studies indicating that diatoms may be natural hybrids [42,47]. For example, new bioinformatics tools will be needed to detect cases of mono-allelic gene expression or allele exclusion to fully understand their contribution to transcriptional regulation of genes and subsequent phenotypic variation, particularly in natural environments. The advent of technologies to assess the extent of genetic and epigenetic phenomena in driving diatom adaptations to changing environments over evolutionarily-relevant timescales will be equally critical for assessing the fate of diatoms in a future ocean modified by human-induced climate change.
Acknowledgements We acknowledge funding from the European Research Council, Louis D Foundation, Gordon and Betty Moore Foundation, Oceanomics to CB and the National Centre for Scientific Research to LT. AR was funded by a MEMO-LIFE International PhD Fellowship Program. We thank Atsuko Tanaka for the images presented in Figure 1.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.pbi.2017.02.001.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Lazarus D, Barron J, Renaudie J, Diver P, Turke A: Cenozoic planktonic marine diatom diversity and correlation to climate change. PLoS One 2014, 9:e84857.
2.
Kooistra WH, Medlin LK: Evolution of the diatoms (Bacillariophyta). IV. A reconstruction of their age from small subunit rRNA coding regions and the fossil record. Mol Phylogenet Evol 1996, 6:391-407.
3.
Keeling PJ: The endosymbiotic origin, diversification and fate of plastids. Philos Trans R Soc Lond B Biol Sci 2010, 365:729-748.
4.
Gould SB, Waller RF, McFadden GI: Plastid evolution. Annu Rev Plant Biol 2008, 59:491-517.
5.
Field CB, Behrenfeld MJ, Randerson JT, Falkowski P: Primary production of the biosphere: integrating terrestrial and oceanic components. Science 1998, 281:237-240.
6.
Patil S, Moeys S, von Dassow P, Huysman MJ, Mapleson D, De Veylder L, Sanges R, Vyverman W, Montresor M, Ferrante MI: Identification of the meiotic toolkit in diatoms and exploration of meiosis-specific SPO11 and RAD51 homologs in the sexual species Pseudo-nitzschia multistriata and Seminavis robusta. BMC Genom 2015, 16:930.
7.
Huysman MJ, Martens C, Vandepoele K, Gillard J, Rayko E, Heijde M, Bowler C, Inze D, Van de Peer Y, De Veylder L et al.: Genome-wide analysis of the diatom cell cycle unveils a novel type of cyclins involved in environmental signaling. Genome Biol 2010, 11:R17. www.sciencedirect.com
Diatom genomics and epigenomics Tirichine, Rastogi and Bowler 53
8.
Zofall M, Grewal SI: Swi6/HP1 recruits a JmjC domain protein to facilitate transcription of heterochromatic repeats. Mol Cell 2006, 22:681-692.
9.
Falkowski PG: Evolution of the nitrogen cycle and its influence on the biological sequestration of CO2 in the ocean. Nature 1997, 387:272-275.
10. Treguer PJ, De La Rocha CL: The world ocean silica cycle. Ann Rev Mar Sci 2013, 5:477-501. 11. Katz MEF, Finkel ZV, Grzebyk D, Knoll AH, Falkowski PG: Evolutionary trajectories and biogeochemical impacts of marine eukaryotic phytoplankton. Annu Rev Ecol Evol Syst 2004, 35:523-556. 12. Martin KR: The chemistry of silica and its potential health benefits. J Nutr Health Aging 2007, 11:94-97. 13. Wachter H, Lechleitner M, Artner-Dworzak E, Hausen A, Jarosch E, Widner B, Patsch J, Pfeiffer K, Fuchs D: Diatomaceous earth lowers blood cholesterol concentrations. Eur J Med Res 1998, 3:211-215. 14. Danil de Namor AF, El Gamouz A, Frangie S, Martinez V, Valiente L, Webb OA: Turning the volume down on heavy metals using tuned diatomite. A review of diatomite and modified diatomite for the extraction of heavy metals from water. J Hazard Mater 2012, 241–242:14-31. 15. Kroger N, Poulsen N: Diatoms-from cell wall biogenesis to nanotechnology. Annu Rev Genet 2008, 42:83-107. 16. Tanaka T, Maeda Y, Veluchamy A, Tanaka M, Abida H, Marechal E, Bowler C, Muto M, Sunaga Y, Tanaka M et al.: Oil accumulation by the oleaginous diatom Fistulifera solaris as revealed by the genome and transcriptome. Plant Cell 2015, 27:162-176. Sequencing of an oleaginous pennate diatom revealed an allodiploid genome structure with an unusual pattern of expression which might be due to the simultaneous growth and oil accumulation. 17. Raposo MF, de Morais RM, Bernardo de Morais AM: Bioactivity and applications of sulphated polysaccharides from marine microalgae. Mar Drugs 2013, 11:233-252. 18. Specht EA, Mayfield SP: Algae-based oral recombinant vaccines. Front Microbiol 2014, 5:60.
Evolution and metabolic significance of the urea cycle in photosynthetic diatoms. Nature 2011, 473:203-207. The authors report in diatoms genetic evidence for the significance of ornithine urea cycle which is absent from green algae and plants. 27. Montsant A, Jabbari K, Maheswari U, Bowler C: Comparative genomics of the pennate diatom Phaeodactylum tricornutum. Plant Physiol 2005, 137:500-513. 28. Rayko E, Maumus F, Maheswari U, Jabbari K, Bowler C: Transcription factor families inferred from genome sequences of photosynthetic stramenopiles. New Phytol 2016, 188:52-66. 29. Fortunato AE, Jaubert M, Enomoto G, Bouly JP, Raniello R, Thaler M, Malviya S, Bernardes JS, Rappaport F, Gentili B et al.: Diatom phytochromes reveal the existence of far-red-lightbased sensing in the ocean. Plant Cell 2016, 28:616-628. The authors demonstrated using a genetic approach the presence of red/ far red light sensing in diatoms mediated by phytochromes. 30. Maumus F, Allen AE, Mhiri C, Hu H, Jabbari K, Vardi A, Grandbastien MA, Bowler C: Potential impact of stress activated retrotransposons on genome evolution in a marine diatom. BMC Genom 2009, 10:624. 31. Galachyants YP, Zakharova YR, Petrova DP, Morozov AA, Sidorov IA, Marchenkov AM, Logacheva MD, Markelov ML, Khabudaev KV, Likhoshway YV et al.: Sequencing of the complete genome of an araphid pennate diatom Synedra acus subsp. radians from Lake Baikal. Dokl Biochem Biophys 2015, 461:84-88. 32. Keeling PJ, Burki F, Wilcox HM, Allam B, Allen EE, Amaral Zettler LA, Armbrust EV, Archibald JM, Bharti AK, Bell CJ et al.: The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing. PLoS Biol 2014, 12:e1001889. A community effort which led to the creation of a single database with more than 650 assembled and functionally annotated genomes from a range of protists. 33. Karsenti E, Acinas SG, Bork P, Bowler C, De Vargas C, Raes J, Sullivan M, Arendt D, Benzoni F, Claverie JM et al.: A holistic approach to marine eco-systems biology. PLoS Biol 2011, 9:e1001177.
19. Nazmi A, Hauck R, Davis A, Hildebrand M, Corbeil LB, Gallardo RA: Diatoms and diatomaceous earth as novel poultry vaccine adjuvants Poult Sci.
34. Allen AE, Laroche J, Maheswari U, Lommer M, Schauer N, Lopez PJ, Finazzi G, Fernie AR, Bowler C: Whole-cell response of the pennate diatom Phaeodactylum tricornutum to iron starvation. Proc Natl Acad Sci U S A 2008, 105:10438-10443.
20. Bowler C, Allen AE, Badger JH, Grimwood J, Jabbari K, Kuo A, Maheswari U, Martens C, Maumus F, Otillar RP et al.: The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 2008, 456:239-244. Genome sequencing of the first pennate diatom revealed an important rate of gene diversification compared to yeast and metazoans as well as expansion of gene families and presence of genes of bacterial origin.
35. Morrissey J, Sutak R, Paz-Yepes J, Tanaka A, Moustafa A, Veluchamy A, Thomas Y, Botebol H, Bouget FY, McQuaid JB et al.: A novel protein, ubiquitous in marine phytoplankton, concentrates iron at the cell surface and facilitates uptake. Curr Biol 2015, 25:364-371.
21. Moustafa A, Beszteri B, Maier UG, Bowler C, Valentin K, Bhattacharya D: Genomic footprints of a cryptic plastid endosymbiosis in diatoms. Science 2009, 324:1724-1726. Evidence for green genes in diatoms. 22. Armbrust EV, Berges JA, Bowler C, Green BR, Martinez D, Putnam NH, Zhou S, Allen AE, Apt KE, Bechner M et al.: The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 2004, 306:79-86. This paper describes genome structure and features of the first centric diatom to be sequenced. 23. Liu H, Fu Y, Li B, Yu X, Xie J, Cheng J, Ghabrial SA, Li G, Yi X, Jiang D: Widespread horizontal gene transfer from circular single-stranded DNA viruses to eukaryotic genomes. BMC Evol Biol 2011, 11:276. 24. Armbrust EV: The life of diatoms in the world’s oceans. Nature 2009, 459:185-192. 25. Bowler C, Vardi A, Allen AE: Oceanographic and biogeochemical insights from diatom genomes. Ann Rev Mar Sci 2010, 2:333-365. 26. Allen AE, Dupont CL, Obornik M, Horak A, Nunes-Nesi A, McCrow JP, Zheng H, Johnson DA, Hu H, Fernie AR et al.: www.sciencedirect.com
36. Montsant AA, Coesel AE, De Martino S, Falciatore A, Mangogna A, Siaut M, Heijde M, Jabbari M, Maheswari K, Rayko U et al.: Identification and comparative genomic analysis of signaling and regulatory components in the diatom Thalassiosira pseudonana. J Phycol 2007, 43:585-604. 37. Malviya S, Scalco E, Audic S, Vincent F, Veluchamy A, Poulain J, Wincker P, Iudicone D, de Vargas C, Bittner L et al.: Insights into global diatom distribution and diversity in the world’s ocean. Proc Natl Acad Sci U S A 2016:E1516-E1525. 38. Lima-Mendez G, Faust K, Henry N, Decelle J, Colin S, Carcillo F, Chaffron S, Ignacio-Espinosa JC, Roux S, Vincent F et al.: Ocean plankton. Determinants of community structure in the global plankton interactome. Science 2015, 348:1262073. 39. Finlay BJ: Global dispersal of free-living microbial eukaryote species. Science 2002, 296:1061-1063. 40. d’Ovidio F, De Monte S, Alvain S, Dandonneau Y, Levy M: Fluid dynamical niches of phytoplankton types. Proc Natl Acad Sci U S A 2010, 107:18366-18370. 41. Sjoqvist C, Godhe A, Jonsson PR, Sundqvist L, Kremp A: Local adaptation and oceanographic connectivity patterns explain genetic differentiation of a marine diatom across the North Sea-Baltic Sea salinity gradient. Mol Ecol 2015, 24:2871-2885. Current Opinion in Plant Biology 2017, 36:46–55
54 Genome studies and molecular genetics
42. Rynearson TA, Armbrust EV: Maintenance of clonal diversity during a spring bloom of the centric diatom Ditylum brightwellii. Mol Ecol 2005, 14:1631-1640. Molecular evidence for hybridization in diatoms. 43. Casteleyn G, Leliaert F, Backeljau T, Debeer AE, Kotaki Y, Rhodes L, Lundholm N, Sabbe K, Vyverman W: Limits to gene flow in a cosmopolitan marine planktonic diatom. Proc Natl Acad Sci U S A 2010, 107:12952-12957. 44. Chen GR: Genetically distinct populations of a diatom co-exist during the North Atlantic spring bloom. Limnol Oceanogr 2016, 61(6):2165-2179 http://dx.doi.org/10.1002/lno.10361. 45. Casteleyn G, Adams NG, Vanormelingen P, Debeer AE, Sabbe K, Vyverman W: cast(Bacillariophyceae): genetic and morphological evidence. Protist 2009, 160:343-354. 46. D’Alelio D, Amato A, Kooistra WH, Procaccini G, Casotti R, Montresor M: Internal transcribed spacer polymorphism in Pseudo-nitzschia multistriata (Bacillariophyceae) in the Gulf of Naples: recent divergence or intraspecific hybridization? Protist 2009, 160:9-20. 47. Casteleyn G, Adams NG, Vanormelingen P, Debeer AE, Sabbe K, Vyverman W: Natural hybrids in the marine diatom Pseudonitzschia pungens (Bacillariophyceae): genetic and morphological evidence. Protist 2009, 160:343-354. 48. Hart MC, Green DH, Bresnan E, Bolch CJ: Large subunit ribosomal RNA gene variation and sequence heterogeneity of Dinophysis (Dinophyceae) species from Scottish coastal waters. Harmful Algae 2007, 6:271-287. 49. Edvardsen B, Shalchian-Tabrizi K, Jakobsen KS, Medlin LK, Dahl E, Brubak S, Paasche E: Genetic variability and molecular phylogeny of Dinophysis species (Dinophyceae) from Norwegian waters inferred from single cell analyses of rDNA. J Phycol 2003, 39:395-408. 50. Keverne B: Monoallelic gene expression and mammalian evolution. Bioessays 2009, 31:1318-1326. 51. Shykind BM: Regulation of odorant receptors: one allele at a time. Hum Mol Genet 2005, 14 Spec No 1:R33-R39. 52. De Riso V, Raniello R, Maumus F, Rogato A, Bowler C, Falciatore A: Gene silencing in the marine diatom Phaeodactylum tricornutum. Nucleic Acids Res 2009, 37:e96. 53. Daboussi F, Leduc S, Marechal A, Dubois G, Guyot V, PerezMichaut C, Amato A, Falciatore A, Juillerat A, Beurdeley M et al.: Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology. Nat Commun 2014, 5:3831. 54. Hamilton ML, Haslam RP, Napier JA, Sayanova O: Metabolic engineering of Phaeodactylum tricornutum for the enhanced accumulation of omega-3 long chain polyunsaturated fatty acids. Metab Eng 2014, 22:3-9. 55. Niu YF, Wang X, Hu DX, Balamurugan S, Li DW, Yang WD, Liu JS, Li HY: Molecular characterization of a glycerol-3-phosphate acyltransferase reveals key features essential for triacylglycerol production in Phaeodactylum tricornutum. Biotechnol Biofuels 2016, 9:60. 56. Falciatore A, Casotti R, Leblanc C, Abrescia C, Bowler C: Transformation of nonselectable reporter genes in marine diatoms. Mar Biotechnol (N Y) 1999, 1:239-251. 57. Karas BJ, Diner RE, Lefebvre SC, McQuaid J, Phillips AP, Noddings CM, Brunson JK, Valas RE, Deerinck TJ, Jablanovic J et al.: Designer diatom episomes delivered by bacterial conjugation. Nat Commun 2015, 6:6925. The authors report a conjugation-based protocol for P. tricornutum transformation based on transfer of plamsids from Escherichia coli to diatoms. 58. Niu YF, Yang ZK, Zhang MH, Zhu CC, Yang WD, Liu JS, Li HY: Transformation of diatom Phaeodactylum tricornutum by electroporation and establishment of inducible selection marker. Biotechniques 2012, 52. 59. Miyahara M, Aoi M, Inoue-Kashino N, Kashino Y, Ifuku K: Highly efficient transformation of the diatom Phaeodactylum Current Opinion in Plant Biology 2017, 36:46–55
tricornutum by multi-pulse electroporation. Biosci Biotechnol Biochem 2013, 77:874-876. 60. Kaur S, Spillane C: Reduction in carotenoid levels in the marine diatom Phaeodactylum tricornutum by artificial microRNAs targeted against the endogenous phytoene synthase gene. Mar Biotechnol (N Y) 2015, 17:1-7. 61. Nymark M, Sharma AK, Sparstad T, Bones AM, Winge P: A CRISPR/Cas9 system adapted for gene editing in marine algae. Sci Rep 2016, 6:24951. 62. Maheswari U, Jabbari K, Petit JL, Porcel BM, Allen AE, Cadoret JP, De Martino A, Heijde M, Kaas R, La Roche J et al.: Digital expression profiling of novel diatom transcripts provides insight into their biological functions. Genome Biol 2010, 11: R85. 63. Bailleul B, Berne N, Murik O, Petroutsos D, Prihoda J, Tanaka A, Villanova V, Bligny R, Flori S, Falconet D et al.: Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms. Nature 2015, 524:366-369. Evidence for energetic exchanges between the plastid and mitochondria necessary for carbon fixation and growth in diatoms. 64. Huysman MJ, Fortunato AE, Matthijs M, Costa BS, Vanderhaeghen R, Van den Daele H, Sachse M, Inze D, Bowler C, Kroth PG et al.: AUREOCHROME1a-mediated induction of the diatom-specific cyclin dsCYC2 controls the onset of cell division in diatoms (Phaeodactylum tricornutum). Plant Cell 2013, 25:215-228. 65. Maumus F, Rabinowicz P, Bowler C, Rivarola M: Stemming epigenetics in marine stramenopiles. Curr Genom 2011, 12:357-370. 66. Rastogi A, Lin X, Lombard B, Loew D, Tirichine L: Probing the evolutionary history of epigenetic mechanisms: what can we learn from marine diatoms. AIMS Genet 2015, 2:173-191. 67. Veluchamy A, Lin X, Maumus F, Rivarola M, Bhavsar J, Creasy T, O’Brien K, Sengamalay NA, Tallon LJ, Smith AD et al.: Insights into the role of DNA methylation in diatoms by genome-wide profiling in Phaeodactylum tricornutum. Nat Commun 2013, 4. 68. Huff JT, Zilberman D: Dnmt1-independent CG methylation contributes to nucleosome positioning in diverse eukaryotes. Cell 2014, 156:1286-1297. The authors found a novel DNA methylatransferase that fulfils the function of animal dnmt1 methylating DNA in a GC context with clustered methylation over nucleosome linkers, contributing to nucleosome positionning between clusters. 69. Rastogi A, Murik O, Bowler C, Tirichine L: PhytoCRISP-Ex: a web-based and stand-alone application to find specific target sequences for CRISPR/CAS editing. BMC Bioinf 2016, 17:261. The authors built a user friendly software to search for target sequences in 13 phytoplankton species. 70. Traller JC, Cokus SJ, Lopez DA, Gaidarenko O, Smith SR, McCrow JP, Gallaher SD, Podell S, Thompson M, Cook O et al.: Genome and methylome of the oleaginous diatom Cyclotella cryptica reveal genetic flexibility toward a high lipid phenotype. Biotechnol Biofuels 2016, 9:258. 71. Rogato A, Richard H, Sarazin A, Voss B, Cheminant Navarro S, Champeimont R, Navarro L, Carbone A, Hess WR, Falciatore A: The diversity of small non-coding RNAs in the diatom Phaeodactylum tricornutum. BMC Genom 2014, 15:698. 72. Huang A, He L, Wang G: Identification and characterization of microRNAs from Phaeodactylum tricornutum by highthroughput sequencing and bioinformatics analysis. BMC Genom 2011, 12:337. 73. Norden-Krichmar TM, Allen AE, Gaasterland T, Hildebrand M: Characterization of the small RNA transcriptome of the diatom, Thalassiosira pseudonana. PLoS One 2011, 6:e22870. 74. Lopez-Gomollon S, Beckers M, Rathjen T, Moxon S, Maumus F, Mohorianu I, Moulton V, Dalmay T, Mock T: Global discovery and characterization of small non-coding RNAs in marine microalgae. BMC Genom 2014, 15:697. www.sciencedirect.com
Diatom genomics and epigenomics Tirichine, Rastogi and Bowler 55
75. Cruz de Carvalho MH, Sun HX, Bowler C, Chua NH: Noncoding and coding transcriptome responses of a marine diatom to phosphate fluctuations. New Phytol 2016, 210:497-510.
81. Roy RS, Price DC, Schliep A, Cai G, Korobeynikov A, Yoon HS, Yang EC, Bhattacharya D: Single cell genome analysis of an uncultured heterotrophic stramenopile. Sci Rep 2014, 4:4780.
76. Lin X, Tirichine L, Bowler C: Protocol: chromatin immunoprecipitation (ChIP) methodology to investigate histone modifications in two model diatom species. Plant Methods 2012, 8:48.
82. Riemann L, Steward GF, Azam F: Dynamics of bacterial community composition and activity during a mesocosm diatom bloom. Appl Environ Microbiol 2000, 66:578-587.
77. Veluchamy A, Rastogi A, Lin X, Lombard B, Murik O, Thomas Y, Dingli F, Rivarola M, Ott S, Liu X et al.: An integrative analysis of post-translational histone modifications in the marine diatom Phaeodactylum tricornutum. Genome Biol 2015, 16:102. The authors decribe for the first time the identification of several PTMs in a diatom and draw a chromatin landscape demonstrating the presence of a histone code sharing features with animals and plants and others unique to diatoms. 78. Vire E, Brenner C, Deplus R, Blanchon L, Fraga M, Didelot C, Morey L, Van Eynde A, Bernard D, Vanderwinden JM et al.: The Polycomb group protein EZH2 directly controls DNA methylation. Nature 2006, 439:871-874. 79. Ringrose L, Ehret H, Paro R: Distinct contributions of histone H3 lysine 9 and 27 methylation to locus-specific stability of polycomb complexes. Mol Cell 2004, 16:641-653. 80. Bhattacharya D, Price DC, Yoon HS, Yang EC, Poulton NJ, Andersen RA, Das SP: Single cell genome analysis supports a link between phagotrophy and primary plastid endosymbiosis. Sci Rep 2012, 2:356.
www.sciencedirect.com
83. Leblanc K, Arı´stegui J, Armand J, Assmy P, Beker B, Bode B, Breton E, Cornet V, Gibson J, Gosselin MP et al.: A global diatom database—abundance, biovolume and biomass in the world ocean. Earth Syst Sci Data 2012, 4:149-165. 84. Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987, 4:406-425. 85. Guillou L, Bachar D, Audic S, Bass D, Berney C, Bittner L, Boutte C, Burgaud G, de Vargas C, Decelle J et al.: The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote small subunit rRNA sequences with curated taxonomy. Nucleic Acids Res 2013, 41:D597-604. 86. Felsenstein J: Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985, 39:783-791. 87. Tamura K, Nei M, Kumar S: Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci U S A 2004, 101:11030-11035. 88. Kumar S, Stecher G, Tamura K: MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 2016, 33:1870-1874.
Current Opinion in Plant Biology 2017, 36:46–55