Diatoms are phototrophic, unicellular, and eukaryotic organisms. They originate from secondary endosymbiosis, a specific evolutionary process. Accordingly, their cells and organelles have a typical organisation, as revealed by ultrastructural investigations. Diatoms possess specific compartments and structures, including a silica shell surrounding the diatom cell, so-called silica deposition vesicles (SDVs), as well as complex plastids that are surrounded by four membranes.

Here we provide an overview of diatom organelles, and recapitulate recent information obtained from 3D imaging of whole diatom cells, focusing on the subcellular topology of the model diatom Phaeodactylum tricornutum. This chapter will not discuss issues of the cell wall and the SDVs, which are covered in chapters 13 and 14.

of the primary ocean productivity [START_REF] Field | Primary production of the biosphere: integrating terrestrial and oceanic components[END_REF], are at the basis of food webs and of the biological CO2 pump, i.e. the main carbon storage pathway in the oceans [START_REF] Field | Primary production of the biosphere: integrating terrestrial and oceanic components[END_REF][START_REF] Smetacek | Role of sinking in diatom life-history cycles: ecological, evolutionary and geological significance[END_REF]. These diverse microalgae can reach up to 100.000 species [START_REF] Malviya | Insights into global diatom distribution and diversity in the world's ocean[END_REF] thriving in different freshwater and ocean environments, including the ice [START_REF] Thomas | Antarctic sea ice--a habitat for extremophiles[END_REF] and turbulent waters [START_REF] Margalef | Life-forms of phytoplankton as survival alternatives in an unstable environment[END_REF].

A possible reason for their ecological success is their high metabolic plasticity [START_REF] Falciatore | Diatom molecular research comes of age: model species for studying phytoplankton biology and diversity[END_REF], which is illustrated, e.g. by the presence of chimeric pathways [START_REF] Allen | Evolution and metabolic significance of the urea cycle in photosynthetic diatoms[END_REF], peculiar photoprotective responses [START_REF] Bailleul | An atypical member of the lightharvesting complex stress-related protein family modulates diatom responses to light[END_REF]) and specific bioenergetic features [START_REF] Bailleul | Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms[END_REF].

While genomic studies [START_REF] Armbrust | The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism[END_REF][START_REF] Bowler | The Phaeodactylum genome reveals the evolutionary history of diatom genomes[END_REF]) and large scale genomic approaches [START_REF] Malviya | Insights into global diatom distribution and diversity in the world's ocean[END_REF]) have provided relevant insights on diatom evolution and biodiversity, we still miss comprehensive knowledge on the diatoms cell biology. This is a critical aspect, since previous work has suggested that, for instance, the physiology and metabolism of diatoms are determined by their peculiar cell organisations and more particularly by the morphology and arrangement of key energy-producing organelles machineries, such as the plastids and mitochondria [START_REF] Bailleul | Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms[END_REF][START_REF] Flori | Plastid thylakoid architecture optimizes photosynthesis in diatoms[END_REF]).

In the following chapter, we first introduce the structure and function of important diatom subcellular compartments, namely nucleus, endoplasmic reticulum, Golgi apparatus, endosomes, vacuoles, peroxisomes, autophagosomes, cytoplasmic membranes, mitochondria, and plastids. Next, we illustrate recent advancement in understanding the subcellular architecture of a diatom cell (focusing on the plastid and its interaction with other organelles), based on 3D imaging techniques in the model species Phaeodactylum tricornutum. Finally, we discuss a particular case of symbiosis involving diatoms and dinoflagellates (Dinotoms), which are widely distributed in both freshwater and marine environments, displaying different levels (temporary or permanent) of interaction between the two organisms.

The cell nucleus. The nucleus of diatoms is surrounded by a typical nuclear envelope (just like in other eukaryotes), in which nuclear pores are visible in ultrastructural investigations (e.g. [START_REF] Round | Diatoms: biology and morphology of the genera[END_REF]). Within the nucleus, heterochromatic and euchromatic areas including, one or more nucleoli, are present. The nucleus can be visualised by dyes inserting into the genomic DNA such as DAPI (e.g. [START_REF] Tanaka | Ultrastructure and membrane traffic during cell division in the marine pennate diatom Phaeodactylum tricornutum[END_REF]) or Hoechst 33342 [START_REF] Schellenberger Costa | Aureochrome 1a is involved in the photoacclimation of the diatom Phaeodactylum tricornutum[END_REF]. The outer membrane of the nuclear envelope is not only connected to the endoplasmic reticulum (ER) membrane, but also continuous with the outermost membrane of the plastid (Fig. 1a, 1d, see also scheme in Fig. 1c), which has been termed chloroplast ER (cER) membrane (see below).

This cellular arrangement is difficult to visualize in ultrastructural studies with respect to the ER-nuclear envelope connection. However, it has been revealed by localisation studies of ER proteins fused to GFP, in which the ER lumen and the lumen of the nuclear envelope are marked (e.g. [START_REF] Apt | In vivo characterization of diatom multipartite plastid targeting signals[END_REF]).

With respect to nuclear division, early investigations indicated the presence of microtubule-organizing centres (MTOCs) and at least a partial breakdown of the nuclear envelope in some diatoms [START_REF] Manton | Observations on the Fine Structure and Development of the Spindle at Mitosis and Meiosis in a Marine Centric Diatom (Lithodesmium Undulatum): II. The Early Meiotic Stages in Male Gametogenesis[END_REF][START_REF] Tippit | Mitosis in the pinnate diatom surirella ovalis[END_REF][START_REF] Tippit | Cell division in two large pennate diatoms Hantzschia and Nitzschia III. A new proposal for kinetochore function during prometaphase[END_REF]. However, a more recent investigation of the diatom P. tricornutum surprisingly showed that in the course of nuclear division, at least one nuclear membrane is maintained [START_REF] Tanaka | Ultrastructure and membrane traffic during cell division in the marine pennate diatom Phaeodactylum tricornutum[END_REF]. In addition, no evidence was obtained in this study for a mitotic apparatus that includes microtubules. Instead, an ingrowing furrow is involved in the penetration and bisection of the nucleus. Import of many proteins into the nucleus seems to be equivalent to the canonical pathway of eukaryotes, in which proteins are exposing a nuclear localisation signal (NLS) that allows import with the help of importins. Protein targeting into the nucleus has been demonstrated for the aureochrome photoreceptor proteins [START_REF] Schellenberger Costa | Aureochrome 1a is involved in the photoacclimation of the diatom Phaeodactylum tricornutum[END_REF].

Endoplasmic reticulum. The endoplasmic reticulum (ER) is composed of one highly branched cisterna that forms a network covering nearly all cytoplasmic regions (as shown by decoration with GFP fusion proteins, (e.g. [START_REF] Apt | In vivo characterization of diatom multipartite plastid targeting signals[END_REF]). Also, the ER-Tracker Blue-White DPX (Invitrogen) was recently applied for visualizing the ER of the diatom P. tricornutum [START_REF] Shao | Comparative characterization of putative chitin deacetylases from Phaeodactylum tricornutum and Thalassiosira pseudonana highlights the potential for distinct chitin-based metabolic processes in diatoms[END_REF]. Beside its biochemical functions such as lipid synthesis [START_REF] Zulu | Current trends to comprehend lipid metabolism in diatoms[END_REF], the ER is the platform for synthesis of proteins of the endomembrane systems, of the cytoplasmic membrane, as well as of secreted proteins. Soluble proteins that either are restrained in the ER lumen or that are directed from the ER to other organelles, as well as some membrane proteins, possess an Nterminal signal peptide (SP), which is predicted by algorithms like SignalP (for the version 5.0 of SignalP see [START_REF] Armenteros | SignalP 5.0 improves signal peptide predictions using deep neural networks[END_REF]. In other eukaryotes, several proteins, not possessing a SP, can also be integrated into a membrane either by an SRP-iNDependent (SND) targeting pathway (e.g. [START_REF] Aviram | The SND proteins constitute an alternative targeting route to the endoplasmic reticulum[END_REF])), or in case of proteins with only one C-terminal transmembrane domain, by a C-tail insertion pathway [START_REF] Chio | Mechanisms of tail-anchored membrane protein targeting and insertion[END_REF][START_REF] Moog | Higher complexity requires higher accuracy: tail-anchored protein targeting to the outer envelope membrane of plant plastids via a specific cterminal motif[END_REF]. The presence of both mechanisms in diatoms still has to be investigated in detail.

N-linked protein glycosylation, which is an important function of the ER, has been studied in diatoms (e.g. [START_REF] Baïet | N-glycans of Phaeodactylum tricornutum diatom and functional characterization of its N-acetylglucosaminyltransferase I enzyme[END_REF][START_REF] Mathieu-Rivet | N-and O-Glycosylation Pathways in the Microalgae Polyphyletic Group[END_REF]).

Like in other eukaryotes, transport of proteins from the ER to the Golgi apparatus and vice versa is supposed to take place via COPII and COPI vesicles, respectively. Soluble proteins of the ER lumen, targeted to the Golgi apparatus, have to be retranslocated to the ER. For this step a C-terminal signal, a so-called KDEL signal, is required. In case of the BiP proteins of P. tricornutum and T. pseudonana a C-terminal DDEL tetrapeptide might acts as an "ER retention signal" (e.g. [START_REF] Apt | In vivo characterization of diatom multipartite plastid targeting signals[END_REF][START_REF] Sheppard | Characterization of an endoplasmic reticulum-associated silaffin kinase from the diatom Thalassiosira pseudonana[END_REF]).

Golgi apparatus. The Golgi apparatus is an important organelle involved in several biochemical functions, including sorting of proteins with a post-Golgi destination. This organelle, as in other cells, can be identified on the ultrastructural level by its wellpronounced stacks of disk-like compartments called cisternae (Fig. 1,2). Vesicles supposed to traffic from or to the Golgi apparatus were identified in the surroundings of this organelle in P. tricornutum (e.g. [START_REF] Tanaka | Ultrastructure and membrane traffic during cell division in the marine pennate diatom Phaeodactylum tricornutum[END_REF]).

A recent study proposes the Golgi apparatus of P. tricornutum to be a single stack of cisternae, positioned near the cell nucleus ( [START_REF] Tanaka | Ultrastructure and membrane traffic during cell division in the marine pennate diatom Phaeodactylum tricornutum[END_REF], Fig. 1a,2a). With regard to the cell cycle, the Golgi apparatus in P. tricornutum duplicates prior to showing that (at least) two Golgi stacks per cell can occur; P, Plastid; Nu, Nucleus; G, Golgi apparatus; bar 1 µm. b Detail of putative multivesicular bodies (MVBs). Bar: 100 nm. c Immunogold labeling of (heterologously) overexpressed GFP-tagged Pex16 protein, showing peroxisomal localisation (arrow) in P. tricornutum (primary antibody α-GFP 1:1000; secondary antibody (rabbit anti goat) coupled to ultrasmall gold 1:100; silver enhancement 15 min). Bar: 500 nm.

karyokinesis [START_REF] Tanaka | Ultrastructure and membrane traffic during cell division in the marine pennate diatom Phaeodactylum tricornutum[END_REF], Fig. 2a), so that both daughter cells are equipped with a Golgi apparatus just after cytokinesis. The Golgi apparatus of diatoms can form complicated arrangements with other compartments. In some diatoms the Golgi apparatus has a tight association with the ER and the mitochondria (so-called Golgi-ER-Mitochondrion unit, G-ER-M unit), or it can encircle the nucleus forming a perinuclear shell (see: [START_REF] Medlin | Evolution of the diatoms: V. Morphological and cytological support for the major clades and a taxonomic revision[END_REF][START_REF] Schmid | The special Golgi-ER-mitochondrium unit in the diatom genus Coscinodiscus[END_REF]).

With the Golgi-Tracker Red (Beyotime, Beijing, China), a specific dye is available for visualizing the Golgi apparatus in live diatom cells [START_REF] Shao | Comparative characterization of putative chitin deacetylases from Phaeodactylum tricornutum and Thalassiosira pseudonana highlights the potential for distinct chitin-based metabolic processes in diatoms[END_REF]. Additionally, several proteins can be used as specific Golgi markers. For sub-compartmental localisation, a predicted β1,2-Xylosyltranferase (XylT) can be used to specifically label the Golgi cisternae [START_REF] Liu | Addressing various compartments of the diatom model organism Phaeodactylum tricornutum via sub-cellular marker proteins[END_REF]. This protein partially overlaps in its localisation with a putative retromer subunit (Vps29), indicating different functions in different cisternae of the Golgi apparatus of P. tricornutum [START_REF] Liu | Addressing various compartments of the diatom model organism Phaeodactylum tricornutum via sub-cellular marker proteins[END_REF]. Also, the GDP-Lfucose transporter and the fucosyltransferase (FuT) are useful Golgi-stack specific markers in P. tricornutum [START_REF] Zhang | Characterization of a GDP-Fucose Transporter and a Fucosyltransferase Involved in the Fucosylation of Glycoproteins in the Diatom Phaeodactylum tricornutum[END_REF]. Finally, the Rab-family small GTPase Sec4 might be involved in the formation of post-Golgi vesicles [START_REF] Tanaka | Ultrastructure and membrane traffic during cell division in the marine pennate diatom Phaeodactylum tricornutum[END_REF].

Endosomes. Endosomes are important organelles of eukaryotic cells as their functions include the transport of endocytosed material to organelles involved in molecule degradation (lysosome, vacuole), the recycling of proteins to be transported to the cytoplasmic membrane/extracellular space after endocytosis and directing newly synthesised proteins to a degradation organelle. Diatom endosomes are not well studied. So far, several genes encoding endosomal proteins including Rab5 were identified in the genome of P. tricornutum [START_REF] Zheng | Molecular exploration of algal interaction between the diatom Phaeodactylum tricornutum and the dinoflagellate Alexandrium tamarense[END_REF]. To compare diatoms and land plants, it would be of interest to test if the trans-Golgi network (TGN) and the early endosome (EE) have overlapping functions, as is the case in higher plants (e.g. [START_REF] Dettmer | Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis[END_REF][START_REF] Viotti | Endocytic and secretory traffic in Arabidopsis merge in the trans-Golgi network/early endosome, an independent and highly dynamic organelle[END_REF]). Similarly, the presence of late endosomes/multivesicular bodies (MVBs), the latter having characteristic internal vesicles, are not well characterised in diatoms. One example for a putative MVB is shown in Fig. 2b.

Vacuole. Like most plant and algal cells, diatoms also harbour a vacuole. An enlargement of the vacuole volume was observed in a nitrate reductase knockout strain of P. tricornutum, propagated in NO3 --containing medium [START_REF] Mccarthy | Nitrate reductase knockout uncouples nitrate transport from nitrate assimilation and drives repartitioning of carbon flux in a model pennate diatom[END_REF] and in WT cells under phosphate limitations [START_REF] Dell'aquila | Mobilization and cellular distribution of phosphate in the diatom Phaeodactylum tricornutum[END_REF]. One of the pronounced functions of the vacuole is the storage of chrysolaminarin, a noncrystalline ß-glucan that is used by diatoms as carbohydrate storage. Several reports focusing on chrysolaminarin synthesis and degradation indicated that a central glucan synthase is located in the tonoplast membrane and utilises UDP glucose [START_REF] Roessler | UDPglucose pyrophosphorylase activity in the diatom cyclotella cryptica. Pathway of chrysolaminarin biosynthesis[END_REF]) on the cytoplasmic side, secreting chrysolaminarin as a linear molecule into the vacuole [START_REF] Hildebrand | Effects of chrysolaminarin synthase knockdown in the diatom Thalassiosira pseudonana: Implications of reduced carbohydrate storage relative to green algae[END_REF][START_REF] Huang | Reduced vacuolar β-1, 3-glucan synthesis affects carbohydrate metabolism as well as plastid homeostasis and structure in Phaeodactylum tricornutum[END_REF]. Furthermore, three transglycosylases have been identified in P. tricornutum that reside in the tonoplast membrane [START_REF] Huang | Diatom Vacuolar 1, 6-β-Transglycosylases can Functionally Complement the Respective Yeast Mutants[END_REF]. They possess N-terminal signal peptides and single transmembrane helices at the C-termini, followed by a short domain that may represent cytoplasmic tails. Accordingly, the enzymatically active moieties are supposed to be located on the vacuolar side of the tonoplast, where they may add carbohydrate side chains to the chrysolaminarin chain [START_REF] Huang | Diatom Vacuolar 1, 6-β-Transglycosylases can Functionally Complement the Respective Yeast Mutants[END_REF].

The tonoplast, the vacuolar membrane in diatoms, can be stained with the dye MDY-64 [START_REF] Huang | Diatom Vacuolar 1, 6-β-Transglycosylases can Functionally Complement the Respective Yeast Mutants[END_REF], and several tonoplast integrated proteins, involved in important functions of the vacuole including carbohydrate metabolism, were localised (e.g. [START_REF] Huang | Diatom Vacuolar 1, 6-β-Transglycosylases can Functionally Complement the Respective Yeast Mutants[END_REF][START_REF] Schreiber | The central vacuole of the diatom Phaeodactylum tricornutum: identification of new vacuolar membrane proteins and of a functional di-leucine-based targeting motif[END_REF]).

Two different targeting routes of proteins to the vacuole have been identified in higher plants. In the "canonical" route, newly synthesised vacuolar proteins first traffic via the secretory system to the trans Golgi/early endosome, where they are sorted for final targeting to the vacuole. Some vacuolar proteins are targeted on a second route from the ER to the vacuole, bypassing the Golgi apparatus (e.g. [START_REF] Viotti | The endoplasmic reticulum is the main membrane source for biogenesis of the lytic vacuole in Arabidopsis[END_REF]).

Currently, we do not know if the latter route is present in diatoms, while there are strong indications for the existence of the "canonical" targeting route.

In the case of vacuolar 1,6-β-transglycosylases putative tyrosine-based and di-leucine based motifs were identified [START_REF] Huang | Diatom Vacuolar 1, 6-β-Transglycosylases can Functionally Complement the Respective Yeast Mutants[END_REF], which might act as targeting signals.

Consistent with this assumption is the finding that a di-leucine motif has been shown to be essential for correct targeting of tonoplast proteins in P. tricornutum [START_REF] Schreiber | The central vacuole of the diatom Phaeodactylum tricornutum: identification of new vacuolar membrane proteins and of a functional di-leucine-based targeting motif[END_REF].

Peroxisomes. Peroxisomes are single membrane-bound organelles, which harbour a variety of pathways, including fatty acid degradation and detoxification of reactive oxygen species (ROS). Along with the majority of eukaryotes, diatoms also possess peroxisomes, as documented by both in silico and in vivo studies [START_REF] Winkler | Isolation and characterization of peroxisomes from diatoms[END_REF]. Genes encoding typical peroxisomal biogenesis proteins, so-called peroxins (Pex) have been identified in centric and pennate diatoms [START_REF] Armbrust | The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism[END_REF][START_REF] Gabaldón | Peroxisomes in parasitic protists[END_REF][START_REF] Gentekaki | Extreme genome diversity in the hyperprevalent parasitic eukaryote Blastocystis[END_REF][START_REF] Gonzalez | A single peroxisomal targeting signal mediates matrix protein import in diatoms[END_REF]Mix et al. 2018). Most detailed information about peroxisome biology in diatoms exists for P. tricornutum. In this species, not only a comprehensive set of peroxins is encoded in the genome, but also several enzymes for peroxisomal metabolism [START_REF] Gonzalez | A single peroxisomal targeting signal mediates matrix protein import in diatoms[END_REF]. Using confocal and electron microscopy, many of these proteins were localised as GFP fusion proteins in P. tricornutum peroxisomes, which in electron microscopy appear as round, single membrane bound compartments usually smaller than 500 nm (Fig. 2c) (Mix et al. 2018). Moreover, protein (co)localisation in diatom peroxisomes can be studied using additional fluorescent markers such as YFP, CFP and mRuby3 Among the peroxisomal proteins identified in P. tricornutum are a catalase for detoxification of reactive oxygen species, enzymes of the glyoxylate cycle, and a complete set of enzymes for fatty acid beta-oxidation [START_REF] Gonzalez | A single peroxisomal targeting signal mediates matrix protein import in diatoms[END_REF]). These findings indicate that diatom peroxisomes contain the typical metabolic functions known form other eukaryotes. It is unknown yet whether there are unexpected biochemical pathways or reactions in diatom peroxisomes.

One peculiarity of diatom peroxisomes is the lack of one of the two classical import pathways for peroxisomal matrix proteins: P. tricornutum peroxisomal proteins contain peroxisomal targeting signals of type 1 (PTS1), a C-terminal tripeptide usually composed of the amino acids SKL (and derivatives of this motif). The less common PTS2 (an N-terminal nonapeptide) seems to play no role in peroxisomal protein import in diatoms [START_REF] Gonzalez | A single peroxisomal targeting signal mediates matrix protein import in diatoms[END_REF]. This was concluded from the complete absence of peroxisomal proteins with PTS2 targeting signals, and by the missing PTS2 receptor (Pex7) and Pex7-binding motif in the PTS2 co-receptor (Pex5) of diatoms [START_REF] Gonzalez | A single peroxisomal targeting signal mediates matrix protein import in diatoms[END_REF]Mix et al. 2018). However, the lack of the PTS2 import pathway apparently is not a specific feature of diatom peroxisomes, and is found in other eukaryotes including Drosophila, Caenorhabditis elegans, Cyanidioschyzon merolae and haptophytes (see [START_REF] Gonzalez | A single peroxisomal targeting signal mediates matrix protein import in diatoms[END_REF]Mix et al. 2018) and references therein).

Autophagosomes. Autophagosomes, organelles involved in delivering cytoplasmic

regions and/or organelles to the lysosome/vacuole, are hard to detect. Their occurrence under stress conditions and their role in clearance of defect organelles has been predicted by the expression of genes that encode putative autophagosomal proteins in T. pseudonana and P. tricornutum [START_REF] Rastogi | Integrative analysis of large scale transcriptome data draws a comprehensive landscape of Phaeodactylum tricornutum genome and evolutionary origin of diatoms[END_REF][START_REF] Shemi | Elucidating the composition and conservation of the autophagy pathway in photosynthetic eukaryotes[END_REF].

Cytoplasmic membrane. The cytoplasmic membrane surrounds the cytoplasm and is involved in many cellular functions. Several reports are available dealing with the import and export of molecules, catalysed by proteins integrated into the cytoplasmic membrane, e.g. aquaporins, a bicarbonate transporter (SLC4) and the metal ion transporters ZIP (zinc transporter protein) (e.g. [START_REF] Coale | Reduction-dependent siderophore assimilation in a model pennate diatom[END_REF][START_REF] Kazamia | Endocytosis-mediated siderophore uptake as a strategy for Fe acquisition in diatoms[END_REF][START_REF] Matsui | Plasma membrane-type aquaporins from marine diatoms function as CO2/NH3 channels and provide photoprotection[END_REF][START_REF] Nakajima | SLC4 family transporters in a marine diatom directly pump bicarbonate from seawater[END_REF]. Another example of a cytoplasmic membrane protein is the plasma membrane intrinsic protein 1 [START_REF] Liu | Addressing various compartments of the diatom model organism Phaeodactylum tricornutum via sub-cellular marker proteins[END_REF]. Furthermore, the so-called PDZ proteins (PDZ1, PDZ2), were found to be located in the plasma membrane. Tagging with eGFP has shown that cytoplasmic membrane proteins are not always evenly distributed as it is the case for PDZ2, but can be also enriched in defined spots in case of the eGFP fused PDZ1 protein [START_REF] Liu | Addressing various compartments of the diatom model organism Phaeodactylum tricornutum via sub-cellular marker proteins[END_REF]. Membrane integrated receptors are essential for signalling at the cytoplasmic membrane. Genes and transcripts of G-protein coupled receptors were identified recently [START_REF] Mojib | Comparative transcriptomics supports the presence of G protein-coupled receptor-based signaling in unicellular marine eukaryotes[END_REF], and may indicate the presence of signalling pathways in diatoms, although an in-depth characterisation is still pending.

The existence of endocytosis is suggested by staining the cytoplasmic membrane with the dye FM4-64 (e.g. [START_REF] Liu | Addressing various compartments of the diatom model organism Phaeodactylum tricornutum via sub-cellular marker proteins[END_REF]) and additionally by application of endocytosis inhibitors in case of siderophore uptake studies [START_REF] Kazamia | Endocytosis-mediated siderophore uptake as a strategy for Fe acquisition in diatoms[END_REF]. Very recently, internalisation of partially condensed silicic acid (oligosilicates) via endocytosis was proposed [START_REF] Annenkov | The probable mechanism for silicon capture by diatom algae: assimilation of polycarbonic acids with diatoms-is endocytosis a key stage in building of siliceous frustules?[END_REF]. In addition, an endocytosis motif (D/ExxxL) is present in case of the iron starvation-induced protein 1 (ISIP1), involved in the uptake of siderophores via endocytosis. This motif is known to interact with the adaptor protein-2 (AP-2) complex [START_REF] Kazamia | Endocytosis-mediated siderophore uptake as a strategy for Fe acquisition in diatoms[END_REF][START_REF] Lommer | Genome and low-iron response of an oceanic diatom adapted to chronic iron limitation[END_REF]. Thus, clathrin-mediated endocytosis seems to occur in diatoms.

Mitochondria. The mitochondria of diatoms vary in shape and quantities (e.g. [START_REF] Schober | Organelle studies and proteome analyses of mitochondria and plastids fractions from the diatom Thalassiosira pseudonana[END_REF]). Typically, diatom mitochondria show either an oval or tubularlike morphology, with sizes range from approximately 0.5 to 1 µm in diameter [START_REF] Schober | Organelle studies and proteome analyses of mitochondria and plastids fractions from the diatom Thalassiosira pseudonana[END_REF][START_REF] Werner | The biology of diatoms[END_REF]. Like in other eukaryotes, mitochondria of diatoms possess two membranes, the innermost membrane forming tubular cristae. The genome in the diatom mitochondrial matrix has a size between 35 to 80 kbp, encoding typically less than 40 proteins (e.g. [START_REF] Guillory | Recurrent loss, horizontal transfer, and the obscure origins of mitochondrial introns in diatoms (Bacillariophyta)[END_REF]). In a P. tricornutum cell one large, branched mitochondrion, winding along the plastid, can be found per cell (see 3D model in Fig. 1).

For specific localisation studies, "mitotracker" dyes (MitoTracker Orange or MitoTracker Green FM, Invitrogen, USA) were used (e.g. [START_REF] Marter | Optimized mRuby3 is a Suitable Fluorescent Protein for in vivo Co-localization Studies with GFP in the Diatom Phaeodactylum tricornutum[END_REF][START_REF] Tanaka | Ultrastructure and membrane traffic during cell division in the marine pennate diatom Phaeodactylum tricornutum[END_REF]). They are of special interest for co-localisation of mitochondrial proteins fused with a fluorescent protein, but also for studying mitochondrial dynamics under different physiological conditions or with respect to the cell cycle (e.g. [START_REF] Tanaka | Ultrastructure and membrane traffic during cell division in the marine pennate diatom Phaeodactylum tricornutum[END_REF]). A protocol for the isolation of a high-quality mitochondrial fraction for diatoms was recently published for Thalassiosira pseudonana [START_REF] Schober | Organelle studies and proteome analyses of mitochondria and plastids fractions from the diatom Thalassiosira pseudonana[END_REF]. Proteome studies [START_REF] Schober | Organelle studies and proteome analyses of mitochondria and plastids fractions from the diatom Thalassiosira pseudonana[END_REF]) might contribute considerably to the physiological and evolutionary understanding of mitochondria in diatoms.

Nucleus-encoded proteins transported into the mitochondrial matrix of diatoms are equipped with a matrix targeting signal at their N-terminus. These targeting sequences can be predicted according to [START_REF] Gruber | Deducing intracellular distributions of metabolic pathways from genomic data[END_REF][START_REF] Gruber | Intracellular metabolic pathway distribution in diatoms and tools for genome-enabled experimental diatom research[END_REF], and be used for screening diatom genomes for mitochondrial matrix proteins in general. In parallel, identification of a matrix targeting signal in a protein of interest can lead to in silico localisation predictions, as it was shown, e.g., for a contiguous translational fusion encoding an N-terminal triosephosphate isomerase (TPI) and a C-terminal GAPDH of the diatom P. tricornutum [START_REF] Liaud | Compartment-specific isoforms of TPI and GAPDH are imported into diatom mitochondria as a fusion protein: evidence in favor of a mitochondrial origin of the eukaryotic glycolytic pathway[END_REF]. Other targeting signals such as those for proteins of the outer and inner membrane have to be identified and studied in detail. In the genome of P. tricornutum, several genes were identified encoding proteins known to be essential for protein import into mitochondria. These include factors for the TOM-and TIM-complex in addition to components of the SAM-complex and for inner membrane targeting [START_REF] Delage | In silico survey of the mitochondrial protein uptake and maturation systems in the brown alga Ectocarpus siliculosus[END_REF]. Thus, there is striking evidence that in diatoms similar mitochondrial protein import mechanisms operate as in other eukaryotes.

Plastid. Whereas the organelles described above are present in nearly all eukaryotes (for exceptions regarding of peroxisomes in certain chromalveolates, see e.g. (Mix et al. 2018;[START_REF] Moog | Genomic and proteomic evidence for the presence of a peroxisome in the apicomplexan parasite Toxoplasma gondii and other Coccidia[END_REF])), the plastid types found in diatoms are restricted to most organisms of the supergroup Chromalveolata (Cavalier-Smith 1999). Although the numbers and sizes of complex plastids vary in diatoms [START_REF] Round | Diatoms: biology and morphology of the genera[END_REF], they all contain four surrounding membranes, at variance with plastids with two surrounding membranes that are present in green and red alga, glaucocystophytes and land plants.

Complex plastids of diatoms originated in the course of a higher order endosymbiosis (e.g. [START_REF] Keeling | Chromalveolates and the Evolution of Plastids by Secondary Endosymbiosis 1[END_REF]), in which a red alga was engulfed by another eukaryote and reduced to a complex plastid (e.g. [START_REF] Cavalier-Smith | Membrane heredity and early chloroplast evolution[END_REF][START_REF] Douglas | Cryptomonad algae are evolutionary chimaeras of two phylogenetically distinct unicellular eukaryotes[END_REF]).

Already in 1998, a protocol for the isolation of plastids from the diatoms Odontella sinensis (Greville) Grunow and Coscinodiscus granii Gough was published [START_REF] Wittpoth | Functional characterization of isolated plastids from two marine diatoms[END_REF]. These plastid fractions were photosynthetically active and would be a highly suitable resource for studying the protein composition of diatom plastids.

However, for neither diatom are genome data available yet. To overcome this limitation, a plastid isolation protocol for the model diatom T. pseudonana was published recently [START_REF] Schober | Organelle studies and proteome analyses of mitochondria and plastids fractions from the diatom Thalassiosira pseudonana[END_REF]. The herewith-isolated plastid fraction was used for extensive proteome maps for both diatom plastid and mitochondria, though lacking stromal plastid proteins.

Of the four membranes of diatom plastids, the outermost membrane (chloroplast endoplasmic reticulum membrane, cERM) is facing the cytoplasm and believed to be the remnant of a phagotrophic membrane, which surrounded the red alga symbiont in the early stages of the evolution of complex plastids (e.g. [START_REF] Cavalier-Smith | Membrane heredity and early chloroplast evolution[END_REF]). The second outermost membrane (membrane II), also termed the periplastid membrane (PPM), might be the remnant of the cytoplasmic membrane of the engulfed red alga, whereas the two innermost membranes (membrane III is also called outer Envelope Membrane oEM and IV, i.e. the inner Envelope Membrane iEM) are homologous to the plastid envelope membranes of the red alga symbiont.

In diatoms, the outermost membrane of the plastid is connected to the outer nuclear membrane (Fig. 1d, see also scheme in Fig. 1c). Therefore, the lumen between the outermost plastid membrane and the PPM, and the lumen of the host ER form a continuous space, although both have different functions. The adjacent lumen, which is delimited by the PPM and membrane III, is called periplastid compartment (PPC), and is considered to be the remnant of the cytoplasm of the engulfed red alga.

The diatom stroma harbours a plastid genome ranging from approximately 100 up to 200 kbp in different species (e.g. [START_REF] Yu | Evolution of the plastid genomes in diatoms[END_REF])) as well as 70S-type ribosomes.

Finally, the stromal membrane system is typically composed of a so-called 'girdle lamella', which is a triple thylakoid structure encircling the central thylakoids. The central thylakoids form a network of internal membranes typically enriched in galactolipids and contain the photosynthetic complexes devoted to photosynthetic electron transfer. In diatoms, these membranes have different topologies than in vascular plants and green algae (see below).

New interpretations of older ultrastructural data (see e.g. [START_REF] Gibbs | The route of entry of cytoplasmically synthesized proteins into chloroplasts of algae possessing chloroplast ER[END_REF])), discussed and summarised in (Cavalier-Smith 2018) as well as the results from a newer study [START_REF] Flori | Ultrastructure of the periplastidial compartment of the diatom Phaeodactylum tricornutum[END_REF] prompt again speculations of an additional compartment within the PPC, the "periplastid reticulum" (PR) (see Fig. 1d). The PR is surrounded by one membrane and one of its function might be delivery of proteins into the PPC for further sorting (Cavalier-Smith 2018). However, it has still to be shown that the PR is an independent compartment within in the PPC and not an invagination of the PPM [START_REF] Zauner | The Known, the New, and a Possible Surprise: A Re-Evaluation of the Nucleomorph-Encoded Proteome of Cryptophytes[END_REF]. The diatom PPC lacks a nucleomorph, a nucleus relic with only a small coding capacity, found in cryptophytes and chlorarachniophytes [START_REF] Zauner | The Known, the New, and a Possible Surprise: A Re-Evaluation of the Nucleomorph-Encoded Proteome of Cryptophytes[END_REF].

Therefore, in diatoms nearly all of the proteins of complex plastids are nucleusencoded and have to be imported from the cytoplasm. Nucleus-encoded proteins delivered to the PPC and the PPM as well as to the stroma are expressed as preproteins with a characteristic targeting signal at the N-terminus [START_REF] Apt | In vivo characterization of diatom multipartite plastid targeting signals[END_REF][START_REF] Gruber | Protein targeting into complex diatom plastids: functional characterisation of a specific targeting motif[END_REF][START_REF] Lang | Protein transport into "complex" diatom plastids utilizes two different targeting signals[END_REF]). This signal is composed of a signal peptide for co-translational ER import followed by a transit peptide-like sequence, together called bipartite targeting sequence (BTS). A BTS is sufficient to import a protein into the complex plastid, as shown for example by GFP targeting experiments [START_REF] Apt | In vivo characterization of diatom multipartite plastid targeting signals[END_REF].

Furthermore, the first amino acid of the transit-peptide like sequence is indicative for the final location of the imported protein. The presence of an aromatic amino acid or a leucine at this position is characteristic for stroma proteins, whereas PPC located proteins show one of the remaining amino acids at the N-terminus of the transit peptide-like sequence [START_REF] Gruber | Protein targeting into complex diatom plastids: functional characterisation of a specific targeting motif[END_REF][START_REF] Kilian | Identification and characterization of a new conserved motif within the presequence of proteins targeted into complex diatom plastids[END_REF]. These targeting sequences are expected to interact with translocons, for which one has to be present in each of the plastid surrounding membranes. The outermost plastid membrane is studded with 80S ribosomes and proteins imported into the complex plastid traverse this membrane via co-translational, Sec61-dependent transport into the lumen between membrane I and the PPM. It is supposed that the signal peptide of the BTS is cleaved off in this lumen, thereby exposing the transit peptide-like sequence for direct or indirect interaction with a translocator in the PPM. In the PPM, an ERAD (endoplasmic reticulum-associated protein degradation)-like machinery was detected [START_REF] Sommer | Der1-mediated preprotein import into the periplastid compartment of chromalveolates?[END_REF], and named symbiont-specific ERAD-like machinery (SELMA) [START_REF] Hempel | New mechanistic insights into pre-protein transport across the second outermost plastid membrane of diatoms[END_REF][START_REF] Stork | Three old and one new: protein import into red algal-derived plastids surrounded by four membranes[END_REF]. This complex still is the only known putative protein translocation machinery in the PPM of diatoms. Several components of the SELMA machinery were identified, which are at least functionally homologous to ERAD factors [START_REF] Lau | Protein-protein interactions indicate composition of a 480 kDa SELMA complex in the second outermost membrane of diatom complex plastids[END_REF][START_REF] Stork | Three old and one new: protein import into red algal-derived plastids surrounded by four membranes[END_REF][START_REF] Stork | Distribution of the SELMA translocon in secondary plastids of red algal origin and predicted uncoupling of ubiquitindependent translocation from degradation[END_REF]. These include as the membrane components homologous to the ERAD components Der1 (sDer1-1, 1-2), an E3 ligase, sUbx, a rhomboid protease, and the soluble proteins sCdc48-1/2, sUfd1 and sNpl4 in the PPC. According to a current model, proteins pass the PPM by the help of the SELMA components. During its import into the PPM, the N-terminal region of the protein is ubiquitinated, which is essential for the final transport into the PPC via Cdc48, Ufd1 and Npl4. After completion, ubiquitin can be eliminated from the transported proteins by the help of a de-ubiquitinase [START_REF] Hempel | New mechanistic insights into pre-protein transport across the second outermost plastid membrane of diatoms[END_REF]).

An alternative import model has been proposed by (Cavalier-Smith 2018), suggesting that protein transport into the PPC starts via vesicles generated at the PPM, which deliver the proteins to the PR, from which they are transported into the PPC via a SELMA complex.

Non-PPC proteins have to be further targeted either into membrane III or IV, into the stroma or the thylakoids. For passing through membrane III, an Omp85 protein was identified, which is homologous to Toc75, the general import pore in the outer plastid membrane of land plants [START_REF] Bullmann | Filling the gap, evolutionarily conserved Omp85 in plastids of chromalveolates[END_REF]. Additionally, Tic components are encoded in the genome of diatoms (e.g. [START_REF] Gruber | Protein targeting into complex diatom plastids: functional characterisation of a specific targeting motif[END_REF])) indicating that a TIC complex should be present in membrane IV to enable protein import into the stroma.

Proteins with a final destination in the thylakoid lumen have to pass an additional membrane. Proteins of this type might use an equivalent to the ΔpH-dependent protein transport system or a Sec-dependent pathway, both known from land plants (see [START_REF] Grouneva | The thylakoid membrane proteome of two marine diatoms outlines both diatom-specific and species-specific features of the photosynthetic machinery[END_REF]. In case of thylakoid membrane proteins, FCP proteins were characterised which require stromal factors as well as GTP for thylakoid membrane integration [START_REF] Lang | Diatom fucoxanthin chlorophyll a/c-binding protein (FCP) and land plant light-harvesting proteins use a similar pathway for thylakoid membrane insertion[END_REF].

Plastid 3D organisation.

As in other microalgae, the diatom plastid is organised in such a way that the electron transport reactions and the CO2-fixing reactions (Calvin cycle) of photosynthesis are segregated in two distinct compartments: the thylakoids and the pyrenoid, respectively.

The thylakoids host the proteins performing the light-phase (electron transport reactions) of photosynthesis. In plants, these membranes form a continuous compartment [START_REF] Mustárdy | Granum revisited. A three-dimensional model-where things fall into place[END_REF][START_REF] Staehelin | Chloroplast structure: from chlorophyll granules to supramolecular architecture of thylakoid membranes[END_REF] characterised by a clear lateral heterogeneity between grana (stacks of thylakoids) and their connecting membranes, the stroma lamellae. No such clear distinction between grana and stroma lamellae is seen in diatoms (Fig. 3a), where membranes are generally organised in stacks of three layers, the 'girdle lamellae', which run parallel to the envelope membranes. Every thylakoid is bounded by a lipid bilayer enclosing an aqueous lumen.

Thylakoids are densely populated in proteins, which occupy up to 70% of the total membrane area [START_REF] Kirchhoff | Molecular architecture of the thylakoid membrane: lipid diffusion space for plastoquinone[END_REF]. In plants, extensive biochemical, fractionation and immunolocalisation studies have shown that the photosystem I (PSI) and the ATPsynthase (ATPase) accumulate preferentially in the stroma exposed membrane (the stroma lamellae and the grana margins) due to steric repulsion between their bulky stromal protrusions [START_REF] Amunts | Functional organization of a plant Photosystem I: evolution of a highly efficient photochemical machine[END_REF][START_REF] Dekker | Supramolecular organization of thylakoid membrane proteins in green plants[END_REF][START_REF] Junge | Torque generation and elastic power transmission in the rotary FOF 1-ATPase[END_REF]. Conversely, the photosystem II (PSII), which has flat stromal surface, is mostly localised in the grana. The cytochrome b6f complex (Cyt b6f), which functionally connects PSI and PSII, has a more homogeneous localisation in these compartments (reviewed in [START_REF] Albertsson | A quantitative model of the domain structure of the photosynthetic membrane[END_REF][START_REF] Kouřil | Supramolecular organization of photosystem II in green plants[END_REF]). Most likely, the main reason for segregating the photosystems in two different domains of the membranes is to prevent a physical contact between them, which would otherwise lead to excitation energy transfer (spillover) from PSII to PSI. Energy spillover may result in unbalanced energy distribution between the photosystems, as reported for cyanobacteria and red algae under particular growth conditions [START_REF] Biggins | Regulation of excitation energy transfer in organisms containing phycobilins[END_REF].

Immunolocalisation experiments in diatoms have shown a topological segregation of PSI and PSII in different thylakoid domains (as in plants) despite the lack of apparent differentiation between the grana and stroma lamellae [START_REF] Flori | Plastid thylakoid architecture optimizes photosynthesis in diatoms[END_REF]. PSI is found mostly within the peripheral membranes of the girdle, while PSII is located within the innermost thylakoid membranes (Fig. 3b). This segregation could be driven by the same bulkiness rules as in plants with 'protruding' PSI molecules being located in nonappressed peripheral membranes and 'flat' PSIIs being found in the core membranes.

Alternatively, the proposed heterogeneous lipid composition of the two thylakoid fractions could drive this segregation, if the core membranes are enriched in lipids that favour PSII stability and function [START_REF] Lepetit | Molecular dynamics of the diatom thylakoid membrane under different light conditions[END_REF]).

3D reconstruction of the plastids has revealed that single thylakoids often bridge the contiguous girdle lamella (Fig. 3a), possibly enabling electron shuttling between photosynthetic complexes of different stacks. Moreover, it was found that the diameter of the thylakoid lumen (7.1 ± 1.5 nm, unpublished data) is two times larger than the size of the soluble electron carrier (cyt c6 ~ 3.3 x 2.3 nm, [START_REF] Akazaki | Crystallization and Structural Analysis of Cytochrome c6 from the Diatom Phaeodactylum tricornutum at 1.5 Å Resolution[END_REF]). This feature should allow for largely unrestricted diffusion of this hydrophilic redox carrier inside the thylakoid membranes, thus enabling electron flow. Overall, the thylakoids topology of diatoms seems to be conceived to optimise their photosynthesis by avoiding unbalanced energy transfer to the photosystems and enhancing photosynthetic electron transfer. The stroma is the compartment where carbon fixation takes place. In most algae, the enzymes required for carbon fixation (i.e. RuBisCO) are located in a specialised region named the pyrenoid [START_REF] Borkhsenious | The intracellular localization of ribulose-1, 5-bisphosphate carboxylase/oxygenase in Chlamydomonas reinhardtii[END_REF][START_REF] Holdsworth | The isolation and partial characterization of the pyrenoid protein of Eremosphaera viridis[END_REF][START_REF] Lacoste-Royal | Immunocytochemical localization of ribulose-1, 5bisphosphate carboxylase in the pyrenoid and thylakoid region of the chloroplast of Chlamydomonas reinhardtii[END_REF]. Different diatoms show different numbers, plastidic locations, and structures of the pyrenoids [START_REF] Bedoshvili | Chloroplast structure of diatoms of different classes[END_REF]).

In P. tricornutum a single pyrenoid is visible, which occupies the centre of the plastid and is crossed by special thylakoids called 'pyrenoid-penetrating thylakoids' [START_REF] Kikutani | Thylakoid luminal θ-carbonic anhydrase critical for growth and photosynthesis in the marine diatom Phaeodactylum tricornutum[END_REF], (Fig. 3a, brown) swelling in the centre of the Rubisco rich compartment (Fig. 3a,yellow).

Besides revealing the topology of photosynthetic membranes, the 3D reconstruction of a P. tricornutum cell has also revealed interesting aspects of organelles (Fig. 1b). The chloroplast and nucleus appear to have physical contacts at specific locations (Fig. 1c) [START_REF] Flori | Plastid thylakoid architecture optimizes photosynthesis in diatoms[END_REF]. This is understandable, because the outermost membrane of the plastid envelope forms a continuum with the outer nuclear membrane in these organisms. Moreover, the inner nuclear membrane and the periplastid membrane (PPM), but also the outer and inner envelope membrane (oEM and iEM) are facing each other [START_REF] Flori | Ultrastructure of the periplastidial compartment of the diatom Phaeodactylum tricornutum[END_REF]. It has been proposed [START_REF] Flori | Plastid thylakoid architecture optimizes photosynthesis in diatoms[END_REF]) that these contacts between the chloroplast and nucleus could facilitate exchanges (e.g. of signalling molecules) between the two organelles. The plastid-nucleus contacts of diatoms are somehow reminiscent of chloroplast evaginations in plants (stromules) that transiently connect to the nucleus for metabolite exchange and retrograde signalling [START_REF] Dietz | Redox-and reactive oxygen speciesdependent signaling into and out of the photosynthesizing chloroplast[END_REF]. This process, largely studied in plants, has recently received some experimental support also in P. tricornutum [START_REF] Lepetit | High light acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool[END_REF].

Signalling between the two compartments could be mediated by the presence of a vesicular network (VN) that is seen close to the nucleus and in connection with both the oEM and the PPM. The proximity between the nucleus and the plastid, and the vesicular system resembling the one seen in P. tricornutum (see also [START_REF] Tanaka | Ultrastructure and membrane traffic during cell division in the marine pennate diatom Phaeodactylum tricornutum[END_REF]) have been reported in other diatom species [START_REF] Bedoshvili | The cell ultrastructure of diatoms-implications for phylogeny? The Transmission Electron Microscope[END_REF][START_REF] Bedoshvili | Chloroplast structure of diatoms of different classes[END_REF], suggesting that nucleus-plastid interactions may be conserved in both pennate and centric diatoms.

Plastid-mitochondria contacts are also seen in P. tricornutum. Mitochondria-plastid juxtaposition results in numerous physical contacts between the two organelles, which are visible in chemically fixed samples [START_REF] Bailleul | Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms[END_REF]. These interactions could facilitate energy exchange to optimise CO2 assimilation. This might be achieved by establishing an ATP flux commensurate to CO2 assimilation in the light [START_REF] Bailleul | Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms[END_REF][START_REF] Kim | Flux balance analysis of primary metabolism in the diatom Phaeodactylum tricornutum[END_REF]. This process likely requires the presence of transporters facilitating the energetic interactions possible between the two organelles. Because diatom plastids are surrounded by four membranes rather than two as in plants, the molecular nature of these transporters could be different from those of plants, reflecting the different phylogenetic origin of these organisms and their complex evolutionary history.

Endosymbiotic Diatoms. Although most diatoms are free-living individuals, there are a few examples of endosymbiotic diatoms, e.g. in dinoflagellates [START_REF] Chesnick | Ribosomal RNA analysis indicates a benthic pennate diatom ancestry for the endosymbionts of the dinoflagellates Peridinium foliaceum and Peridinium balticum (Pyrrhophyta)[END_REF], in foraminifera [START_REF] Bernhard | Benthic foraminifera of dysoxic sediments: chloroplast sequestration and functional morphology[END_REF][START_REF] Lee | The Identification of Diatoms Isolated as Endosymbionts from Larger Foraminifera from the Gulf of Eilat (Red Sea) and the Description of 2 New Species, Fragilaria shiloi sp. nov. and Navicula reissii sp[END_REF][START_REF] Lee | Identification and distribution of endosymbiotic diatoms in larger foraminifera[END_REF], and in flatworms [START_REF] Van Steenkiste | A new case of kleptoplasty in animals: marine flatworms steal functional plastids from diatoms[END_REF]. Heterotrophic dinoflagellates of different groups may take up the cytoplasm of algal cells, including diatoms, and store them in a single membrane vacuole, the symbiosome [START_REF] Tomas | Peridinium balticum (levander) lemmermann, an unusual dinoflagellate with a mesocaryotic and an eucaryotic nucleus[END_REF]. Such a kleptoplasty process may either involve selectively the ingested plastids only, or in other cases can also include some of the other ingested prey organelles, especially mitochondria and nuclei [START_REF] Johnson | Retention of transcriptionally active cryptophyte nuclei by the ciliate Myrionecta rubra[END_REF]). Most of these associations are temporary, but in a specific group of dinoflagellates, the dinotoms, some endosymbiotic diatoms have evolved into permanent stages [START_REF] Tippit | Apparent amitosis in the binucleate dinoflagellate Peridinium balticum[END_REF][START_REF] Yamada | Discovery of a kleptoplastic 'dinotom'dinoflagellate and the unique nuclear dynamics of converting kleptoplastids to permanent plastids[END_REF]. Dinotoms are widely distributed in both freshwater and marine environments. They may possess at least two types of plastids of different origins [START_REF] Dodge | A dinoflagellate with both a mesocaryotic and a eucaryotic nucleus I. Fine structure of the nuclei[END_REF][START_REF] Hehenberger | Evidence for the retention of two evolutionary distinct plastids in dinoflagellates with diatom endosymbionts[END_REF].

Furthermore, there are different stages of integration of the diatom endosymbionts into the host cells, ranging from permanent endosymbionts to temporary endosymbionts as kleptoplastids [START_REF] Yamada | Discovery of a kleptoplastic 'dinotom'dinoflagellate and the unique nuclear dynamics of converting kleptoplastids to permanent plastids[END_REF]). In the latter case, diatoms have to be taken up by the dinoflagellate regularly to keep the dinoflagellate alive. Interestingly, there is a high specificity regarding the diatom species that can be taken up and utilised as photosynthetic endosymbiont by the host cell. A comparison of two closely related dinotoms, Durinskia capensis and D. kwazulunatalensis, recently revealed two different evolutionary stages. While D. capensis is a kleptoplastic protist, keeping its diatoms temporarily for up to 2 months, D. kwazulunatalensis is able to keep several diatoms species permanently [START_REF] Yamada | Discovery of a kleptoplastic 'dinotom'dinoflagellate and the unique nuclear dynamics of converting kleptoplastids to permanent plastids[END_REF].

Conclusions.

Diatoms have been studied for centuries, mostly regarding their taxonomy. Only in the past 25 years, their molecular cell biology and genetics came into focus, revealing a number of unexpected findings. Modern tools in cell biology now allow a better understanding of peculiar processes and structures, like the formation of a silica cell wall, the complex cellular structures like the chloroplast ER, and their unusual photophysiology. Because of the recent progress in developing molecular tools, like for instance genome editing and genetic transformation via bacterial conjugation, diatoms will have an increasing importance as model organisms to study specific functions of organelles in stramenopile algae.

  Diatoms are key phytoplankton players. By converting CO2, sunlight and nutrients into biomass and oxygen, these unicellular phototrophs contribute up to 20%

Fig. 2 . a

 2a Fig. 2. a Ultrastructure of P. tricornutum (after high pressure freezing, freeze substitution and Epon embedding) as seen on 50 nm ultrathin sections by transmission electron microscopy, showing that (at least) two Golgi stacks per cell can occur; P, Plastid; Nu, Nucleus; G, Golgi apparatus; bar 1 µm. b Detail of putative multivesicular bodies (MVBs). Bar: 100 nm. c Immunogold labeling of (heterologously) overexpressed GFP-tagged Pex16 protein, showing peroxisomal localisation (arrow) in P.tricornutum (primary antibody α-GFP 1:1000; secondary antibody (rabbit anti goat) coupled to ultrasmall gold 1:100; silver enhancement 15 min). Bar: 500 nm.

Fig. 3

 3 Fig. 3. a EM picture of a P. tricornutum cell, superimposed to a partial 3D reconstruction of the thylakoid membranes network (green) from FIB-SEM imaging. Single thylakoids connecting the three layers are visible. Pyrenoid-penetrating thylakoids are show in brown. They become swollen in their centre (yellow). Bar: 1 µm. b Cartoon of the thylakoid membranes in P. tricornutum. Complexes are localised either in the external (green) or in the internal membranes (violet). PSI: photosystem I; PSII: photosystem II, cytb6f: cytochrome b6f complex. ATP synth. ATP synthase.

Fig. 2

 2 Fig. 2 (a) Ultrastructure of P. tricornutum (after high pressure freezing, freeze substitution, and Epon embedding) as seen on 50 nm ultrathin sections by transmission electron microscopy, showing that (at least) two Golgi stacks per cell can occur; P, Plastid; Nu, Nucleus; G, Golgi apparatus; bar 1 μm. (b) Detail of putative multivesicular bodies (MVBs). Bar: 100 nm. (c) Immunogold labeling of (heterologously) overexpressed GFP-taggeg Pex16 protein, showing peroxisomal localisatio (arrow) in P. tricornutum (primary antibody α-GFP 1: 1000; secondary antibody (rabbit anti goat) coupled to ultrasmall gold 1:100; silver enhancement 15 min). Bar: 500 nm

Acknowledgements

References Acknowledgements. The authors would like to thank Dr. K. Bolte, University of Marburg, for the electron microscopy images of Fig. 1d, Fig. 1e and Fig. 2a, and Dr. P. Martinez, IRIG Grenoble, for help in making Fig. 3. D.F., P.-H. J. and G.F. acknowledge support from the CEA DRF impulsion FIB-Bio program. U.G.M. and D.M. thank the Deutsche Forschungsgemeinschaft and the SYNMIKRO research center (Marburg) for support. P.G.K. acknowledges support by the Deutsche Forschungsgemeinschaft as well as the Gordon and Betty Moore Foundation (Project GBMF 4966 "DiaEdit"). G.F., D.F. and S.F. also acknowledge funds from the European Research Council (AdG grant ChloroMito, grant number 833184).

Abbreviations

BTS: bipartite targeting sequence