Diatoms, marine genomics, and the marine ecosystem
The contemporary oceans cover about 70% of the Earth‟s surface. Each year, around 50 billion tons of carbon in the form of CO2 are fixed into organic material by the microorganisms populating the upper sunlit portion of the ocean, and each year a similar amount of organic carbon is transferred into marine ecosystems by sinking and grazing (Figure 1.1). Thus, although accounting for only about 1% of the Earth‟s photosynthetic biomass these organisms, commonly referred to as “phytoplankton”, are responsible for about 45% of our planet‟s annual net primary productivity (NPP) (Field, et al.,1998), while the contribution from macroalgae is estimated to be around 1% (Smith, 1981). The term “phytoplankton” describes a diverse, polyphyletic group of mostly single-celled photosynthetic organisms that drift with the currents in marine and fresh waters including cyanobacteria and eukaryotes. In contemporary marine ecosystems, diatoms constitute one of the most successful groups of phytoplankton, responsible for about 40% of marine net primary productivity. In corollary, diatoms are important producers of the oxygen we breathe and form the base of the food chain in marine ecosystems upon which the higher trophic levels depend.
Diatoms (class Bacillariophyceae) are a group of autotrophic single-celled eukaryotic algae found in all kinds of humid environments and open water masses. One of the most characteristic features of diatoms is their ability to generate a hard outer silica-based cell wall called the frustule which consists of two asymmetrical halves assembled like the two overlapping halves of a Petri dish (Figure 1.2), hence the Greek etymology of “diatom”: dia “through” and temein “to cut”; i.e., “cut in half”. The larger of the two halves is called the epitheca and the inner one is called the hypotheca. The frustule has been proposed to constitute a physically strong and chemically inert protective covering because silica cannot be attacked enzymatically.
When a diatom divides to produce two daughter cells, each cell keeps one of the two halves and grows a smaller half within it. Because the frustule cannot grow once it has been laid down, the average size of diatom cells in a population decreases after each division cycle (Figure 1.3). Once such cells reach a certain minimum size, regeneration of the original size typically occurs via sexual reproduction in which male and female gametes combine to produce a diploid auxospore. This expands in size to generate a cell larger than either parent, which then returns to size-diminishing mitotic divisions. Another consequence of size reduction, particularly for pennate diatoms, is that smaller cells may be mistaken for other species because valve outline and proportions may differ from that of larger cells (Figure 1.4).
Frustules are often finely ornamented with pores, processes, spines, and other distinguishing features, and resemble an elegant pattern of lace drawn at the nanometer scale (Figure 1.5). They come in many fascinating and beautiful shapes, whose intricate architecture is faithfully reproduced from generation to generation, implicating a strict genetic control of the process. Actually, the silicon-based pattern design is used by scientists to distinguish one diatom species from another and forms the basis of diatom classification (Round et al., 1990). This astonishing level of precision has rendered diatoms popular with microscopists as early as the nineteenth century because they were used as guides to improve optical microscope resolution. Nowadays, understanding the biochemical process of frustule formation in diatoms is appealing for nanotechnologists because these architectures far exceed the capabilities of human engineering and are accomplished under mild physiological conditions.
Figure 1.3: Schematic overview of mitotic cell cycle and hypovalve formation in a pennate diatom. Following mitosis and cytokinesis, a specialized vesicle known as the silica deposition vesicle (SDV) forms between the nucleus and the plasma membrane, at the position where the new hypovalve will be generated. The SDV elongates into a tube and spreads out perpendicularly to eventually form a huge vesicle along one side of the cell. A new valve is formed within the SDV by the transport of silica, proteins, and polysaccharides into it, and once complete, it is exocytosed from the cell. The two daughter cells can then separate and grow unidirectionally along the cell division axis by the biogenesis of gridle bands, which are also formed within the SDVs. (from Falciatore and Bowler, 2002)
Figure 1.4: (A) Example of cell size reduction in Coscinodiscus (from Armbrust, 2009). (B) Example of change in valve outline and shape with size reduction in Navicula reinhardtii (from M.G. Kelly, H. Bennion, E.J. Cox, B. Goldsmith, J. Jamieson, S. Juggins D.G. Mann & R.J. Telford. 2005. Common freshwater diatoms of Britain and Ireland: an interactive key. Environment Agency, Bristol.
Figure 1.5: Scanning electron micrographs of diatoms (from Bradbury, 2004). (A) Biddulphia reticulata. The whole shell or frustule of a centric diatom showing valves and girdle bands (size bar = 10 micrometres). (B) Diploneis sp. This picture shows two whole pennate diatom frustules in which raphes or slits, valves, and girdle bands can be seen (size bar = 10 micrometres). (C) Eupodiscus radiatus. View of a single valve of a centric diatom (size bar = 20 micrometres) (D) Melosira varians. The frustule of a centric diatom, showing both valves and some girdle bands (size bar = 10 micrometres)
Classification and Habitats
The diatom group is among the most diversified group of photosynthetic eukaryotes with more than 200 living genera and approximately 100,000 extant species (Round et al. 1990). Diatoms are traditionally divided into two orders according to cell symmetry: the centrics which are radially symmetrical and the pennates which are bilaterally symmetrical. Further, some pennate diatoms known as raphid diatoms have a slit (raphe) in the cell wall for movement via the secretion of polysaccharides; the araphid pennates lack this slit and are non-motile. Also, an additional subgrouping of centrics is now recognized, the radial centrics and bi/multi polar centrics. The diatom taxonomy thus distinguishes between the four classes (Kooistra, 2007) (Figure 1.6). It is generally agreed that the pennate diatoms evolved from the centric forms and that the raphid pennates evolved from the araphid pennates due to their order of appearance in the fossil record. Indeed, the siliceous frustules preserve remarkably well, and consequently diatoms have a detailed fossil record.
The first reliable diatom fossil is of a centric from the early Jurassic about 185 Million years ago (Mya) (Sims et al., 2006). However, it is believed that diatoms originated further back in time than their fossil record indicates because of the incompleteness of sampling. The lack of earlier fossil records may also be because the first diatoms lacked robust silica frustules. After the early Jurassic period, diatoms are scarce until the Lower Cretaceous (145-110 Mya) where highly diversified centric diatoms occur in many deposits. The first pennate diatoms, the araphids, appear in fossil records from the Late Cretaceous (87–65 Mya) and the raphid pennate diatoms appear at about 50 Mya (Kooistra and Medlin, 1996). The order in which the four diatom groups appeared is in accordance with that inferred with molecular phylogenies, although the molecular clocks place the origin of diatoms at 250 Mya (Sorhannus et al., 2007).
Nowadays, although being the youngest, the pinnate diatoms are by far the most diversified. They are major components of both benthic and pelagic habitats, having either a planktonic existence or being associated with surfaces. On reefs, pennate diatoms can dominate the communities of protists on surfaces such as macroalgae and rocks. The appearance of the raphe was probably a determining feature that permitted subsequent rapid diversification of pennate diatoms into a range of habitats and although it likely evolved to glide on surfaces in benthic diatoms, many raphid pennates now colonize planktonic environments.
Planktonic diatoms live in the photic zone of the ocean, between the surface layer and the nutrient-rich deep chlorophyll maximum (DCM), and generally prefer well-mixed nutrient-rich waters. Species composition of the surface and DCM layer communities are typically quite distinct (Venrick, 1998). When nutrients become scarce diatoms can sink to great depths where it is thought that they lie dormant until conditions become favorable again for growth. Diatoms have also colonized sea-ice ecosystems where other autotrophs are especially scarce (Figure 1.7) and thus ice diatoms constitute an important component of polar food webs.
Figure 1.6: Phylogenetic relationships among Bacillariophyceae (adapted from a figure courtesy of Dr. Wiebe Kooistra from the Stazione Zoologicale Anton Dorn in Naples, Italy). Phylogenies inferred from SSU rRNA-gene regions of diatoms and their stramenopile relatives reveal that radial centrics are the most ancestral and probably paraphyletic. They gave rise to multipolar centrics, which are also paraphyletic. One of its lineages gave rise to the pennates. Within the latter, araphid pennates are paraphyletic and raphid pennates monophyletic.
Figure 1.7: Polar diatom community. The orange ice is filled with diatoms. The diatoms live in the pore spaces between ice crystals and in salty brine water that is within the ice. Some zooplankton in the ocean water are able to eat diatoms from the bottom of the ice. (from The National Oceanic and Atmospheric Administration (NOAA)
Role of diatoms in biogeochemical cycles
Until the Mesozoic, the dominant phytoplankton were organic-walled organisms (cyanobacteria, green algae, acritarchs, and dinoflagellates). Eukaryotic phytoplankton with mineralized skeletons became dominant during the Mesozoic and account for most contemporary marine primary productivity. These include the coccolithophorids (with calcium carbonate cell walls), silicoflagellates and diatoms (with silica-based cell walls). As a result of gravitational settling, the accelerated sinking rates of such heavy cells contributed enormously to the biogeochemical pump, decreasing CO2 and increasing O2 in the atmosphere, and resulting in carbon sequestration in sediments, creating many of today‟s oil and gas reserves and a significant amount of oxygen. The downward flux of dead mineralized phytoplankton is also essential to transfer organic material and ions to the ecosystem living beneath the photic zone. The abundance of diatoms in modern marine ecosystems is such that they constitute a key component of the biological carbon pump that transports carbon to the ocean interior, largely contributing to the long term sequestration of atmospheric CO2 and supporting much of the marine food chain.
Because they use silica to synthesize their frustules, diatoms are also the most significant consumers of silicic acid dissolved in the ocean. Every atom of silicon entering the ocean has been estimated to be incorporated into 40 successive diatom frustules before sinking to the sea floor (Tréguer et al., 1995). Deposits of diatoms formed over geological time as ancient diatoms died and settled to the bottom have built enormous layers of diatom skeletons known as diatomite, or diatomaceous earth. These deposits are mined for use in toothpastes, paints, filtering agents, and abrasives, as well as explosives.
Nitrogen is an essential nutrient for living organisms in being a constituent of nucleic and amino acids. Most marine eukaryotes and cyanobacteria are able to incorporate inorganic (NO3-, NO2-, NH4+) and organic (urea, amino acids) forms of nitrogen. Diatoms tend to dominate other microalgae in nitrate-rich waters (e.g., upwelling environments), but they are rare in nitrogen-poor open ocean ecosystems where recycled nitrogen (e.g., NH4+) and nitrogen fixation drive production. Although eukaryotic phytoplankton do not fix nitrogen gas, some diatom species can elaborate symbiotic relations with nitrogen-fixing cyanobacteria (Foster and Zehr, 2006).
The oxidizing conditions of the modern oceans result in very low concentrations of dissolved iron and the cellular demand from phytoplankton is often in excess of iron availability. This paradox is though to reflect the fact that cellular machineries depending on iron evolved in the iron-replete Proterozoic (2.5 to 0.5 Mya) reducing oceans. Indeed, the oxygen released over time since the appearance of photosynthesis in cyanobacteria gradually filled chemical sinks of unoxidized iron before oxygen levels began to increase in the atmosphere. Nowadays, iron is supplied to phytoplankton by aeolian transport of dust (Cassar et al., 2007) and possibly by the upwelling of deep water from hydrothermal vents. It is now well accepted that iron depletion is a key limitation of phytoplankton growth and the cause of so-called “photosynthesis deserts” (e.g., polar Southern Ocean) which are defined as high nutrient low chlorophyll (HNLC) regions. Conversely, large-scale open ocean iron fertilization experiments have been shown to trigger phytoplankton blooms, especially of pennate diatoms (Boyd et al., 2000).
The vast majority of characterized eukaryotes can now be assigned to one of five or six supergroups (Keeling et al., 2005) which comprise the Opisthokonta and Amoeboza (often united in the Unikonts), Plantae, Excavata, Chromalveolata, and Rhizaria (Figure 1.8). These supergroups have been established on the basis of molecular and ultrastructural data. Molecular analysis involves mainly the analysis of distances in concatenated multi-gene datasets and the conservation of gene fusions, while ultrastructural data involves characterization of different visible characters such as the shape of the mitochondrial inner membrane (cristae), the presence of a plastid, or the number of flagella.
The Unikonts, which includes animal and fungal kingdoms and related forms, have a single basal flagellum on reproductive cells and flat mitochondrial cristae (most eukaryotes have tubular ones). The unikonts have a triple-gene fusion encoding enzymes for synthesis of the pyrimidine nucleotides (carbamoyl phosphate synthase, dihydroorotase, aspartate carbamoyltransferase) that is lacking in bacteria and bikonts. This must have involved a double fusion, a rare pair of events, which provides the first really compelling support for Amoebozoa being sisters to opisthokonts rather than to bikonts (Stechmann et al., 2003).
Excavata is a major assemblage of heterotrophic single-celled organisms which contains a variety of free-living and symbiotic forms. Many excavates such as diplomonads and parabasalids (e.g., Giardia and Trichomonas) lack classical mitochondria, although most retain a mitochondrial organelle in greatly modified form. Others have mitochondria with tubular, discoidal, or in some cases, laminar cristae. Most excavates have two, four, or more flagella.
The Rhizaria unites a heterogeneous group of flagellates and amoebae including cercomonads, foraminifera, and former members of the polyphyletic radiolaria (Cavalier-Smith, 2002). Cercozoa and foraminifera appear to share a unique insertion in their ubiquitin gene that consolidates the Rhizaria supergroup (Archibald et al., 2003).
The group Plantae arose through endosymbiosis whereby a non-photosynthetic single-celled eukaryote (host) engulfed and retained a free-living photosynthetic cyanobacterium to form a primary symbiotic oxygenic eukaryote more than 1,500 Mya (Figure 1.9A). Over time, the prokaryote was reduced to a double membrane-bound plastid and was vertically transmitted to subsequent generations. Part of this process involved the transfer of hundreds of genes from the cyanobacterium/plastid to the eukaryotic host nucleus via a process known as endosymbiotic gene transfer (EGT) concomitant with reduction of the enslaved cyanobacterium genome to become the plastid genome. Recent analysis also suggests the presence of some chlamydial (obligate intracellular bacteria) genes in both plants and red algae, but not in cyanobacteria, and therefore that a chlamydial endosymbiont may also have imprinted during the early stages of the primary endosymbiosis (Becker et al., 2008).
The photosynthetic Plantae ancestor diversified into three lineages: green algae which include land plants and have chlorophyll a and b pigments, rhodophytes (red algae), and glaucophytes which both have chlorophyll a and phycobilin pigments (Reyes-Prieto et al., 2007). The plastids of glaucophytes retain a cyanobacterial-type peptidoglycan layer that differentiates them from red algae. In the ocean, the dominant unicellular algae derived from a primary endosymbiosis are prasinophytes, which belong to the green lineage.
Soon after the split of rhodophytes and green algae, it is hypothesized that a member of the rhodophyte lineage was engulfed by an unknown heterotroph secondary host giving rise to the pigmented ancestor of the Chromalveolata (Cavalier-Smith, 1999) about 1,300 Mya (Yoon et al. 2004) (Figure 1.9B). This supergroup of secondary endosymbionts was proposed as a parsimonious explanation for the presence of plastids of red algal origin in photosynthetic members of both the Alveolata and Chromista. The Alveolata group is well supported and includes ciliates, dinoflagellates, and apicomplexa. Chromista unifies diverse microbial lineages with red algal plastids (and their non-photosynthetic descendents) including cryptophytes, haptophytes, and heterokonts (which include diatoms).
Over evolutionary time, extant Chromalveolata have lost most of the rhodophyte symbiont structures but the process of secondary endosymbiosis has left its evolutionary signature with the plastid and its unique membrane topography. Secondary plastids are often surrounded by four membranes (in haptophytes, heterokonts, and cryptophytes). The two additional membranes are thought to correspond to the plasma membrane of the engulfed alga and the phagosomal membrane of the host cell. The endosymbiotic acquisition of a eukaryotic cell is represented most clearly in the cryptophytes where the remnant nucleus of the red algal symbiont (the nucleomorph) is present between the inner two and outer two plastid membranes. The retention of the plastid can be attributed to the advantagous autotrophic lifestyle that it confers (many plastid-less cells living today engulf algae for this purpose in a process known as kleptoplastidy).
Besides plastid membrane topology, other evidence also supports the chromalveolate hypothesis which posits that the alveolate group and the less well resolved chromist group acquired secondary red plastids by a single common endosymbiosis. Notably, all photosynthetic chromalveolates contain chlorophyll c, which is absent from other algae (Cavalier-Smith, 1999). Second, two plastid-targeted proteins (glyceraldehyde-3-phosphate dehydrogenase and fructose-1,6-bisphosphate aldolase) have unusual but common features that are unique to chromalveolates (Harper and Keeling, 2003; Patron et al., 2004). However, the chromalveolate hypothesis is hotly debated and may not appear to be monophyletic as originally proposed. Specifically, the inclusion of haptophytes and cryptophytes in Chromalveolata is thus far only weakly supported by phylogenetic analyses using host nucleus-encoded genes (Harper et al., 2005). Further, recent phylogenomic data generated using expressed sequence tags (ESTs) from two species belonging to the supergroup Rhizaria suggest a very robust relationship between Rhizaria and two main clades of the chromalveolate supergroup: stramenopiles and alveolates; while cryptophytes and haptophytes cluster together in a little-supported clade (Burki et al. 2007). The name SAR (Stramenopile, Alveolate, Rhizaria) was proposed to accommodate this new super assemblage of eukaryotes.
At least two scenarios are conceivable to explain the evolution of chlorophyll-c containing plastids. First, a single engulfment of red algae might have occurred at a very early stage of chromalveolate evolution and the resulting plastid was secondarily lost in certain lineages, such as ciliates and Rhizaria. Second, it is possible that stramenopiles, alveolates, or haptophytes and cryptophytes have acquired their secondary plastid through an independent endosymbiosis event. Phylogenomic data incorporating EST data from cryptophytes and haptophytes also support the monophyly of haptophytes and cryptophytes and the association of Rhizaria with alveolates and stramenopiles (Hackett et al., 2007).
In separate, more recent endosymbioses, green algae were independently engulfed by the common ancestor of the chlorarachniophyte amoebae (Rhizaria) and of the euglenids (Excavata), giving rise to two distinct lines of green secondary plastids (Rogers et al., 2007). It has also been postulated that the ancestral dinoflagellate acquired its plastid from a haptophyte through a tertiary endosymbiosis event with plastid replacement (Yoon et al., 2002). Consequently, oxygenic photosynthesis that originated in cyanobacteria about 2,800 Mya has been transferred to a wide diversity of eukaryotes through successive and mixed endosymbiosis events over evolutionary times such that contemporary eukaryotic phytoplankton includes members of all eukaryotic supergroups except the Unikonts (Figure 8).
Table of contents :
Chapter I: Introduction
1.1 Diatoms, marine genomics, and the marine ecosystem
1.1.1 First glance
1.1.2 Diatom biology
1.1.3 Classification and habitats
1.1.4 Role of diatoms in biogeochemical cycles
1.1.5 Evolutionary history
1.1.6 Diatom and (algal) genomics
1.1.7 The diatom Phaeodactylum tricornutum
1.2 Transposable elements
22.214.171.124 Class I elements
126.96.36.199 Class II elements
1.2.3 Impact of TEs on genome evolution
188.8.131.52 The generation of genetic variability in response to stress
184.108.40.206 TE-mediated recombination
220.127.116.11 Gene duplication
Chapter II: Transcription factors in diatom genomes
2.2 Results and Discussion
2.2.1 Transcription factor content in stramenopiles
2.2.2 Transcription factor complement among stramenopiles
2.2.3 Heat Shock Factors (HSFs)
2.2.4 Myb factors
2.2.5 bZIP domain factors
2.2.6 bHLH factors
2.2.7 Expression analysis
2.3 Material and Methods
Chapter III: Potential Impact of Stress Activated Retrotransposons on Genome Evolution in a Marine Diatom
3.2.1 Expansion of LTR Retrotransposons in the P. tricornutum genome
3.2.2 Classification of LTR retrotransposon sequences
3.2.3 Phylogenetic analysis
3.2.4 Expression of LTR retrotransposons in diatoms
3.2.5 Regulation of Blackbeard
3.2.6 Insertion polymorphism between P. tricornutum accessions
3.2.7 Two distinct haplotypes at loci containing TEs
3.2.8 TE-mediated recombination in the P. tricornutum genome
3.2.9 A high diversity of RT domains from micro-planktonic organisms
3.4 Material and Methods
Chapter IV: Epigenetics in P. tricornutum
4.2 Results and Discussion
4.2.1 Histone modifications
18.104.22.168 Histone modifiers
22.214.171.124 Chromatin Extraction and Immunoprecipitation
4.2.2 DNA methylation
126.96.36.199 DNA methyltransferases in diatoms
188.8.131.52 DNA methylation in P. tricornutum
4.2.3 RNA silencing machinery in P. tricornutum
184.108.40.206 Diatom genes putatively involved in RNAi-related processes
220.127.116.11 RNA-directed DNA methylation (RdDM) in P. tricornutum
4.3 Material and Methods
Chapter V: Identification and analysis of transposable elements in the genome of the brown alga Ectocarpus siliculosus
5.2 Results and discussion
5.2.1 Identification of repeated sequences in the E. siliculosus genome
5.2.2 Masking the E. siliculosus genome
5.2.3 Phylogeny of E. siliculosus LTR-retrotransposons
5.2.4 Expression analysis
5.2.5 DNA Methylation in Ectocarpus siliculosus
5.3 Material and Methods
Chapter VI: Conclusions and perspectives
6.1 Conclusions and perspectives