Damien Le Moigne

Nolwenn Guéguen

Juliette Salvaing
email: juliette.salvaing@cea.fr

Lipid Droplets in plants: more than a simple fat storage

Keywords: Lipid Droplets, biotic and abiotic stress, energy storage, lipid remodeling, plants, algae, secondary endosymbiosis

Lipid droplets (LDs) are found in all lineages of living organisms, whether prokaryotes or eukaryotes.

Their structure is rather simple with a core of hydrophobic molecules, mostly Triacylglycerols and Sterol Esters, surrounded by a monolayer of polar lipids with associated proteins. They are constitutively present in storage organs, such as seeds in plants, or adipose tissue in mammals, while they can form in response to various abiotic or biotic stresses in other plant organs and in microalgae. Their biogenesis has been mostly studied in yeast and animals, yet remains poorly understood in plants. In particular, the central role of the photosynthetic plastid in lipid metabolism raises questions about its role in LD biogenesis. Over the years, many functions of LDs have emerged in addition to the obvious role in carbon and energy storage. While some functions appear broadly distributed among organisms and organs, other appear specific to certain tissues and/or lineages.

The present review aims at highlighting specific aspects of LD biogenesis and functions in all photosynthetic organisms, from the historic model plant Arabidopsis thaliana to emerging secondary endosymbiont models such as the diatom Phaeodactylum tricornutum.

Introduction

Lipid Droplets are intracellular lipid inclusions that can be found in very different organisms, and are present in almost all clades composing the tree of life, from Archaea and Bacteria to eukaryotic cells [START_REF] Lundquist | Lipid droplets throughout the evolutionary tree[END_REF]Lupette & Maréchal, 2020;[START_REF] Murphy | The dynamic roles of intracellular lipid droplets : From archaea to mammals[END_REF]. These structures have been observed and described for the first time in the late 19th century [START_REF] Altmann | DIACYLGLYCEROL ACYLTRANSFERASE1 Contributes to Freezing Tolerance[END_REF] and were originally called microsomes or liposomes, but they also have been referred to as lipid bodies, lipid droplets, adiposomes, granules, oleosomes or oil bodies [START_REF] Murphy | The dynamic roles of intracellular lipid droplets : From archaea to mammals[END_REF], depending on the research field, or on the considered organisms or tissues. We will adopt the increasingly used term Lipid Droplets (LDs) in the present review. LDs can be categorized in three different classes depending on their subcellular location [START_REF] Lundquist | Lipid droplets throughout the evolutionary tree[END_REF]. « Cytoplasmic LDs » are present in eukaryote's cytoplasms, « Prokaryotic LDs » in prokaryote's cytoplasms and Plastid LDs, also called plastoglobules, in the plastids of photosynthetic eukaryotes. We will restrict ourselves to the study of Cytoplasmic LDs in eukaryotic photosynthetic organisms, while plastoglobules are discussed in Chapter III.

Photosynthetic organisms are characterized by the presence of a plastid, which can derive from a primary symbiotic event (Archaeplastida that regroup land plants, green, blue and red algae) or from a secondary endosymbiosis event [START_REF] Guéguen | Lipid Droplets in Unicellular Photosynthetic Stramenopiles[END_REF][START_REF] Petroutsos | Evolution of galactoglycerolipid biosynthetic pathways-From cyanobacteria to primary plastids and from primary to secondary plastids[END_REF] (Figure 1). In pluricellular organisms, LDs can be found in various tissues. This great variety is reflected in the composition, biogenesis and main roles of LDs. Many recent reviews [START_REF] Chapman | Mechanisms of lipid droplet biogenesis[END_REF][START_REF] Guéguen | Lipid Droplets in Unicellular Photosynthetic Stramenopiles[END_REF][START_REF] Ischebeck | Lipid droplets in plants and algae : Distribution, formation, turnover and function[END_REF]Leyland, et al., 2020a) have explored the composition and biogenesis pathways of LDs in plants and/or microalgae, of which we give a rapid overview before discussing more extensively the functions of LDs in these organisms.

Lipid Droplets composition and biogenesis

LDs contain a hydrophobic core composed of neutral lipids such as Triacylglycerols (TAGs) and Steryl Esters (SEs) and surrounded by a glycerolipid monolayer surface with associated or embedded proteins, which most often play structural or metabolic roles but can have other functions as we will discuss later [START_REF] Huang | Plant Lipid Droplets and Their Associated Proteins : Potential for Rapid Advances[END_REF] (Figure 2).

Lipid Droplet biogenesis

LD biogenesis is dependent on and starts with the core lipids synthesis. TAG synthesis takes place between the two leaflets of the ER membrane [START_REF] Walther | SENSITIVE TO FREEZING2 Aids in Resilience to Salt and Drought in Freezing-Sensitive Tomato[END_REF]. They can be synthetized de novo by the Kennedy pathway, which is conserved across eukaryotes [START_REF] Chapman | Compartmentation of triacylglycerol accumulation in plants[END_REF].

Briefly, fatty acids (FA) synthetized in the plastid are transported to the ER, where they are esterified with glycerol-3-phospate. Glycerol-3-phosphate acyl transferases (GPATs) catalyse the first esterification on position sn-1 to form lysophosphatidic acid (LPA), then lysophosphatidic acid acyl transferases (LPATs) esterify a second fatty acid on position sn-2 to generate phosphatidic acid (PA).

PA's polar head can be further modified to form other phospholipids such as phosphatidylglycerol (PG) or removed by phosphatidic acid phosphatases (PAPs) to generate diacylglycerol (DAG), the latter serving as a platform for most glycerolipids synthesis. DAG can also be generated by recycling of other glycerolipids through different pathways that are presented on Figure 3. The last reaction to form TAG is then catalysed by Diacylglycerol Acyl Transferases (DGATs), which esterify a third fatty acid on the sn-3 position of DAG. These three successive acyl esterifications are using acyl-CoAs as donor substrates. Alternatively, TAG can be formed directly by transferring a fatty acid from the sn-2 position of a donor glycerolipid (generally Phosphatidylcholine (PC)) to the sn-3 position of DAG, a reaction catalyzed by Phospholipid:Diacylglycerol Acyltransferases (PDATs) (Figure 3). Other core lipids include Sterol Esters (SEs) or wax esters [START_REF] Baud | Seeds as oil factories[END_REF][START_REF] Chapman | Compartmentation of triacylglycerol accumulation in plants[END_REF][START_REF] Ferrer | Emerging roles for conjugated sterols in plants[END_REF] and are also synthesised in the ER membrane of plant cells [START_REF] Ischebeck | Lipid droplets in plants and algae : Distribution, formation, turnover and function[END_REF].

When TAG and/or SE reach a certain concentration, they condense into lenses at specific nucleation sites [START_REF] Choudhary | A conserved family of proteins facilitates nascent lipid droplet budding from the ER[END_REF][START_REF] Thiam | The physics of lipid droplet nucleation, growth and budding[END_REF]. LD nucleation is favoured at points showing higher membrane curvature, especially in ER tubules, and can be modulated by several proteins, in particular Seipin [START_REF] Santinho | Membrane Curvature Catalyzes Lipid Droplet Assembly[END_REF] that can trap TAG and DAG, according to in silico molecular simulations [START_REF] Prasanna | Seipin traps triacylglycerols to facilitate their nanoscale clustering in the endoplasmic reticulum membrane[END_REF][START_REF] Zoni | Seipin accumulates and traps diacylglycerols and triglycerides in its ring-like structure[END_REF]. In yeast, Seipin can associate with PEX30, a protein involved in peroxisome biogenesis, thus defining common nucleation sites for LD and peroxisome [START_REF] Joshi | Lipid droplet and peroxisome biogenesis occur at the same ER subdomains[END_REF][START_REF] Wang | Seipin and the membrane-shaping protein Pex30 cooperate in organelle budding from the endoplasmic reticulum[END_REF]. In mammals, Multiple C2 domain containing transmembrane protein 2 (MCTP2) plays a role similar to that of PEX30 [START_REF] Joshi | Lipid droplet and peroxisome biogenesis occur at the same ER subdomains[END_REF][START_REF] Joshi | Multiple C2 domain-containing transmembrane proteins promote lipid droplet biogenesis and growth at specialized endoplasmic reticulum subdomains[END_REF]. In addition, Seipin can be recruited by LDAF1/Promethin, thus forming a complex that promotes LD formation; LDAF1/Promethin later dissociates from Seipin and covers the surface of LDs [START_REF] Castro | Promethin Is a Conserved Seipin Partner Protein[END_REF][START_REF] Chung | LDAF1 and Seipin Form a Lipid Droplet Assembly Complex[END_REF]. Seipin function in LD biogenesis is conserved in plants [START_REF] Cai | Arabidopsis SEIPIN Proteins Modulate Triacylglycerol Accumulation and Influence Lipid Droplet Proliferation[END_REF] and algae (Lu 2017), although differences are observed. The three Seipin homologs identified in Arabidopsis thaliana have overlapping but not redundant functions [START_REF] Cai | Arabidopsis SEIPIN Proteins Modulate Triacylglycerol Accumulation and Influence Lipid Droplet Proliferation[END_REF][START_REF] Taurino | SEIPIN Proteins Mediate Lipid Droplet Biogenesis to Promote Pollen Transmission and Reduce Seed Dormancy[END_REF]. Seipin 2 and 3, but not Seipin 1, can associate with the vesicle-associated membrane protein (VAMP)-associated protein VAP27, an interaction crucial for LD formation [START_REF] Greer | SEIPIN Isoforms Interact with the Membrane-Tethering Protein VAP27-1 for Lipid Droplet Formation[END_REF].

LDAF1/Promethin homologs, as well have those of FIT2, another important protein in yeast and animals LD biogenesis [START_REF] Choudhary | A conserved family of proteins facilitates nascent lipid droplet budding from the ER[END_REF], have not been identified thus far in plants. However, some specific plant proteins are involved in LD biogenesis such as Lipid Droplet Associated Proteins (LDAPs) [START_REF] Gidda | Lipid Droplet-Associated Proteins (LDAPs) Are Required for the Dynamic Regulation of Neutral Lipid Compartmentation in Plant Cells[END_REF] and the associated LDAP-interacting protein (LDIP) [START_REF] Pyc | Arabidopsis lipid dropletassociated protein (LDAP)-Interacting protein (LDIP) influences lipid droplet size and neutral lipid homeostasis in both leaves and seeds[END_REF], and a recent study has shown that LDIP interacts with Seipin proteins and that this interaction has functional similarities with the Seipin/LDAF1 interaction [START_REF] Pyc | LDIP Cooperates with SEIPIN and LDAP to Facilitate Lipid Droplet Biogenesis in Arabidopsis[END_REF].

The monolayer surrounding LDs likely reflects the membrane composition of the compartment where LD biogenesis occurs. In plants, as well as in non-photosynthetic organisms, it mostly contains phospholipids (Tzen et al., 1993;Tauchi-Sato et al., 2002). However, other classes of lipids have been identified in microalgae. Upon nitrogen starvation, Sulfoquinovosyldiacylglycerol (SQDG) is found in the LDs of the green alga Chlamydomonas rheinardtii [START_REF] Goold | Saturating Light Induces Sustained Accumulation of Oil in Plastidal Lipid Droplets in Chlamydomonas reinhardtii[END_REF][START_REF] Tsai | Dynamics of protein and polar lipid recruitment during lipid droplet assembly in Chlamydomonas reinhardtii[END_REF] and the diatom Phaeodactylum tricornutum [START_REF] Lupette | The architecture of lipid droplets in the diatom Phaeodactylum tricornutum[END_REF], along with a betaine lipid, respectively Mono-and digalactosylglycerol (MGDG and DGDG) are also found in the LD monolayer of Chlamydomonas, especially when algae are submitted to high light stress [START_REF] Goold | Saturating Light Induces Sustained Accumulation of Oil in Plastidal Lipid Droplets in Chlamydomonas reinhardtii[END_REF]. The simultaneous presence of ER lipids (betaine and phospholipids) and plastid lipids (SQDG, MGDG and DGDG) underlines a possible cooperation between the ER and plastid for LD biogenesis in microalgae. Electronic microscopy observations [START_REF] Flori | Ultrastructure of the Periplastidial Compartment of the Diatom Phaeodactylum tricornutum[END_REF][START_REF] Goodson | Structural Correlates of Cytoplasmic and Chloroplast Lipid Body Synthesis in Chlamydomonas reinhardtii and Stimulation of Lipid Body Production with Acetate Boost[END_REF][START_REF] Jaussaud | Stepwise Biogenesis of Subpopulations of Lipid Droplets in Nitrogen Starved Phaeodactylum tricornutum Cells[END_REF][START_REF] Lupette | The architecture of lipid droplets in the diatom Phaeodactylum tricornutum[END_REF] comfort this hypothesis,

1,2-diacylglyceryl-3-O-4'-(N,N,N-trimethyl)-homoserine (DGTS) and 1,2-diacylglyceryl-3-O-2'- (hydroxymethyl)-(N,N,N-trimethyl)-β-alanine (DGTA).
showing tight associations between the plastid and LDs. Interestingly, the role of the chloroplast in LD biogenesis could be conserved in the green tissues of higher plants. Indeed, older studies have shown an association of DGAT with chloroplast envelopes in spinach and Arabisopsis leaves [START_REF] Kaup | A role for diacylglycerol acyltransferase during leaf senescence[END_REF][START_REF] Martin | Subcellular localization of triacylglycerol synthesis in spinach leaves[END_REF], while contacts between LDs and chloroplasts were observed by electron microscopy in aging leaves [START_REF] Brocard | Proteomic Analysis of Lipid Droplets from Arabidopsis Aging Leaves Brings New Insight into Their Biogenesis and Functions[END_REF].

Altogether, the specificities of the proteins involved in LD biogenesis, as well as the increasingly obvious involvement of plastids, underline the interest in further exploring this process in plants, as well as in microalgae where the diversity of the plastid origins and architectures (cf. Figure 1) raises even more questions.

Lipid Droplet associated proteins

Proteins are an essential component of LDs and can be associated with LDs in different ways. Class I proteins are transferred from the RE to the LD and contain a hydrophobic domain forming a hairpin that anchors the protein in the LD (class I). Class II proteins are transferred to the LD surface from the cytosol. Some contain an amphipathic helix that associates with the LD surface while others interact more loosely with LDs through association either with other LD proteins or following posttranslational modifications that add a lipid anchor, such as farsenylation [START_REF] Bersuker | Establishing the lipid droplet proteome : Mechanisms of lipid droplet protein targeting and degradation[END_REF][START_REF] Dhiman | Mechanisms of protein targeting to lipid droplets : A unified cell biological and biophysical perspective[END_REF][START_REF] Olzmann | Dynamics and functions of lipid droplets[END_REF] (Figure 2A). The most abundant proteins at the surface of LDs are often structural proteins, although they can have other functions as will be exposed later in the review. Land plants have four major families of LD proteins, with different tissue specificities and different associated roles [START_REF] Huang | Plant Lipid Droplets and Their Associated Proteins : Potential for Rapid Advances[END_REF] (Figure 2C andD). Oleosins [START_REF] Huang | Oil Bodies and Oleosins in Seeds[END_REF] are the major LD proteins in plant seeds and pollen, but are absent from leaves LDs, which are instead covered with LDAPs [START_REF] Gidda | Lipid Droplet-Associated Proteins (LDAPs) Are Required for the Dynamic Regulation of Neutral Lipid Compartmentation in Plant Cells[END_REF][START_REF] Horn | Identification of a New Class of Lipid Droplet-Associated Proteins in Plants[END_REF]. Oleosins are involved in the regulation of LD size, but also in TAG biosynthesis [START_REF] Parthibane | Oleosin Is Bifunctional Enzyme That Has Both Monoacylglycerol Acyltransferase and Phospholipase Activities[END_REF]. The other two families, Caleosins [START_REF] Naested | Caleosins : Ca2+-binding proteins associated with lipid bodies[END_REF], and Steroleosins [START_REF] Lin | Steroleosin, a Sterol-Binding Dehydrogenase in Seed Oil Bodies[END_REF], are found in all plant tissues.

Oleosins, caleosins and LDAPs homologs could be identified in green algae [START_REF] Huang | Plant Lipid Droplets and Their Associated Proteins : Potential for Rapid Advances[END_REF], but the most abundant LD protein in Chlamydomonas reinhardtii is the Major Lipid Droplet Protein (Moellering & Benning, 2010;[START_REF] Nguyen | Proteomic profiling of oil bodies isolated from the unicellular green microalga Chlamydomonas reinhardtii : With focus on proteins involved in lipid metabolism[END_REF]. Finally, the major LD proteins in secondary endosymbionts show little conservation: in the diatom Phaeodactylum tricornutum, the most abundant LD protein is Stramenopile Lipid Droplet Protein (StLDP) [START_REF] Yoneda | Identification of a Major Lipid Droplet Protein in a Marine Diatom Phaeodactylum tricornutum[END_REF], while it is the Lipid Droplet Surface Protein in other stramenopiles belonging to the Nannochloropsis and Microchloropsis genera [START_REF] Vieler | A Lipid Droplet Protein of Nannochloropsis with Functions Partially Analogous to Plant Oleosins[END_REF]. However, another abundant LD protein in Phaeodactylum called PtLDP1 (Lipid Droplet Protein 1) [START_REF] Wang | A lipid droplet-associated protein involved in lipid droplet biogenesis and triacylglycerol accumulation in the oleaginous microalga Phaeodactylum tricornutum[END_REF] is highly similar to the DOAP1 protein (diatom oleosome-associated protein 1) from another diatom, Fistulifera sp. [START_REF] Guéguen | Lipid Droplets in Unicellular Photosynthetic Stramenopiles[END_REF][START_REF] Nojima | Proteomics Analysis of Oil Body-Associated Proteins in the Oleaginous Diatom[END_REF] (Figure 2B).

Thus, major LD proteins show specificities depending on the considered organism or tissue. Yet, it only gives a glimpse into the proteomic variety found in LDs, which in turns reflects the many roles of this fascinating compartment. Indeed, while energy storage has been the first identified role of LDs, many more have since emerged.

3

Functions for lipid droplets in normal conditions and abiotic stress

Lipid droplets as a source of carbon

In plant seeds, TAGs sored in LDs constitute a reservoir of energy and carbon skeletons to sustain germination and post-germination growth, when the plantlet cannot depend on photosynthesis [START_REF] Graham | Seed Storage Oil Mobilization[END_REF][START_REF] Huang | Oil Bodies and Oleosins in Seeds[END_REF]. While energy storage can rely on other molecules, in particular proteins and starch, TAGs are widely used and can be stored in the embryo itself or in the endosperm [START_REF] Miray | Molecular Control of Oil Metabolism in the Endosperm of Seeds[END_REF]. However, we will not distinguish here between the different tissues that compose the seed. LD mobilization can occur in two ways: lipophagy, a specialized form of autophagy, and lipolysis. A recent study has shown that lipophagy plays a role in LD breakdown during seed germination and post-germination growth in Ricinus communis [START_REF] Han | Genomic Characterization and Expressional Profiles of Autophagy-Related Genes (ATGs) in Oilseed Crop Castor Bean (Ricinus communis L.)[END_REF]). Yet, whether this is specific to certain species or a more general pathway remains to be determined. The most important LD degradation pathway in seeds is lipolysis [START_REF] Graham | Seed Storage Oil Mobilization[END_REF]. It starts by the degradation of Oleosins covering seed LDs by the 26S proteasome [START_REF] Deruyffelaere | Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis[END_REF][START_REF] Deruyffelaere | PUX10 Is a CDC48A Adaptor Protein That Regulates the Extraction of Ubiquitinated Oleosins from Seed Lipid Droplets in Arabidopsis[END_REF][START_REF] Kretzschmar | PUX10 Is a Lipid Droplet-Localized Scaffold Protein That Interacts with CELL DIVISION CYCLE48 and Is Involved in the Degradation of Lipid Droplet Proteins[END_REF]. Then, the TAG lipases SUGAR-DEPENDENT1 (SDP1) and SUGAR-DEPENDENT1-LIKE (SDP1L) [START_REF] Eastmond | SUGAR-DEPENDENT1 Encodes a Patatin Domain Triacylglycerol Lipase That Initiates Storage Oil Breakdown in Germinating Arabidopsis Seeds[END_REF][START_REF] Kelly | Seed Storage Oil Mobilization Is Important But Not Essential for Germination or Seedling Establishment in Arabidopsis[END_REF] catalyze the first step of TAG hydrolysis that ultimately leads to the release of glycerol and free fatty acids. The control of seed LD size, which is dependent on Oleosins [START_REF] Shimada | A novel role for oleosins in freezing tolerance of oilseeds in Arabidopsis thaliana[END_REF][START_REF] Siloto | The Accumulation of Oleosins Determines the Size of Seed Oilbodies in Arabidopsis[END_REF], is crucial to ensure lipolysis efficiency [START_REF] Shimada | A novel role for oleosins in freezing tolerance of oilseeds in Arabidopsis thaliana[END_REF]. Indeed, smaller LDs have a higher surface/volume ratio that facilitates their degradation. Fatty acids can then be converted to acetyl-CoA by -oxidation [START_REF] Goepfert | β-Oxidation in fatty acid degradation and Current[END_REF] in specialized peroxisomes called glyoxysomes [START_REF] Beevers | Microbodies in Higher Plants[END_REF], and used for gluconeogenesis [START_REF] Goepfert | β-Oxidation in fatty acid degradation and Current[END_REF][START_REF] Hayashi | Entering a new era of research on plant peroxisomes[END_REF][START_REF] Miray | Molecular Control of Oil Metabolism in the Endosperm of Seeds[END_REF][START_REF] Quettier | SUGAR-DEPENDENT6 Encodes a Mitochondrial Flavin Adenine Dinucleotide-Dependent Glycerol-3-P Dehydrogenase, Which Is Required for Glycerol Catabolism and Postgerminative Seedling Growth in Arabidopsis[END_REF]. In most albuminous seeds, the TAGs stored in the endosperm are thus converted to sucrose before being transported to the seedling itself [START_REF] Miray | Molecular Control of Oil Metabolism in the Endosperm of Seeds[END_REF].

Alternatively, fatty acids can be reused to build new membrane lipids.

Similarly to what occurs in seeds, the formation of LDs is essential for pollen development as disruption of TAG synthesis leads to pollen abortion [START_REF] Zhang | DGAT1 and PDAT1 Acyltransferases Have Overlapping Functions in Arabidopsis Triacylglycerol Biosynthesis and Are Essential for Normal Pollen and Seed Development[END_REF]. Studies on olive pollen have shown that LD mobilization provides energy for pollen germination [START_REF] Zienkiewicz | New insights into the early steps of oil body mobilization during pollen germination[END_REF].

Moreover, impairment of fatty acid transport to peroxisome [START_REF] Footitt | The COMATOSE ATP-Binding Cassette Transporter Is Required for Full Fertility in Arabidopsis[END_REF], disruption of the TAG lipase OBL1 (Oil body lipase 1) [START_REF] Muller | Characterization of the enzymatic activity and physiological function of the lipid droplet-associated triacylglycerol lipase AtOBL1[END_REF] as well as enlargement of LDs caused by disruption of the three SEIPINs [START_REF] Taurino | SEIPIN Proteins Mediate Lipid Droplet Biogenesis to Promote Pollen Transmission and Reduce Seed Dormancy[END_REF], reduce male fertility. However, the amount of TAGs in LDs is likely insufficient to store enough energy and lipid precursors to sustain the pollen tube growth. Moreover, there is a continuous synthesis of TAGs in this organ, raising the hypothesis that LDs essential role lies elsewhere [START_REF] Ischebeck | Lipids in pollen-They are different[END_REF][START_REF] Ischebeck | Lipid droplets in plants and algae : Distribution, formation, turnover and function[END_REF].

In microalgae, LDs accumulate mainly in response to abiotic stress, in particular deprivation of nutrients, which induces growth arrest. While the photosynthetic apparatus and chloroplast lipids eventually get partially degraded, photosynthesis remains active [START_REF] Abida | Membrane glycerolipid remodeling triggered by nitrogen and phosphorus starvation in Phaeodactylum tricornutum[END_REF][START_REF] Simionato | The Response of Nannochloropsis gaditana to Nitrogen Starvation Includes De Novo Biosynthesis of Triacylglycerols, a Decrease of Chloroplast Galactolipids, and Reorganization of the Photosynthetic Apparatus[END_REF]. In this situation, LDs allow the storage of the excess carbon produced, and provide energy and carbon to quickly restart when the conditions get better. In agreement with this, nitrogen resupply following nitrogen starvation leads to a very rapid degradation of TAGs, as well as other storage components such as starch [START_REF] Mulders | Dynamics of biomass composition and growth during recovery of nitrogen-starved Chromochloris zofingiensis[END_REF][START_REF] Tsai | Recovery from N Deprivation Is a Transcriptionally and Functionally Distinct State in Chlamydomonas[END_REF].

Lipid droplets, lipid synthesis and lipid remodeling

The function of LDs in lipid membrane remodeling and homeostasis has been well described in yeast [START_REF] Graef | Lipid droplet-mediated lipid and protein homeostasis in budding yeast[END_REF][START_REF] Henne | Spatial compartmentalization of lipid droplet biogenesis[END_REF]. Indeed, in Saccharomyces cerevisiae, the KO of the four genes involved in TAG (the DGAT ortholog Dga1p and the PDAT ortholog Lro1), and SE (the acyl-CoA:cholesterol O-acyltransferase orthologs Are1p and Are2p) syntheses severely impairs the yeast ability to respond to stress. Exposure of these mutants to unsaturated free fatty acids (FFA), which are normally taken in and incorporated into TAGs, leads to aberrant membrane formation and ultimately to cell death, suggesting that LD storage of TAGs and SEs acts as a buffer to protect the cell against toxic FFA [START_REF] Petschnigg | Good Fat, Essential Cellular Requirements for Triacylglycerol Synthesis to Maintain Membrane Homeostasis in Yeast[END_REF]. Moreover, growth of these mutants at high temperature in the absence of Inositol leads to severe growth defects and the authors observed a decreased synthesis of membrane phospholipids even in normal growing conditions, suggesting a coordination of membrane lipids and storage lipid syntheses to maintain lipid homeostasis [START_REF] Gaspar | Coordination of Storage Lipid Synthesis and Membrane Biogenesis : EVIDENCE FOR CROSS-TALK BETWEEN TRIACYLGLYCEROL METABOLISM AND PHOSPHATIDYLINOSITOL SYNTHESIS*[END_REF]. In agreement with this idea, a more recent study has shown that LD formation and subsequent degradation by microautophagy are crucial for adaptation of yeast cells to lipid imbalance [START_REF] Vevea | Role for Lipid Droplet Biogenesis and Microlipophagy in Adaptation to Lipid Imbalance in Yeast[END_REF]. The quadruple TAG/SE synthesis mutants also show macroautophagy defects. This may be linked to general ER homeostasis defects or failure to supply the FA necessary for autophagophore formation [START_REF] Henne | Spatial compartmentalization of lipid droplet biogenesis[END_REF][START_REF] Velázquez | Lipid droplet-mediated ER homeostasis regulates autophagy and cell survival during starvation[END_REF], as it is the case for prospore membrane building during sporulation [START_REF] Hsu | Lipid droplets are central organelles for meiosis II progression during yeast sporulation[END_REF]. Finally, a recent study has shown an important function for Lro1, the yeast homolog of PDAT, in the homeostasis of the nuclear envelop and localization of the nucleolus during the cell cycle or in response to stress [START_REF] Barbosa | Compartmentalized Synthesis of Triacylglycerol at the Inner Nuclear Membrane Regulates Nuclear Organization[END_REF].

Lipid Droplets, lipid synthesis and remodeling in microalgae

LD proteomes from green microalgae such as Chlamydomonas reinhardtii [START_REF] Goold | Saturating Light Induces Sustained Accumulation of Oil in Plastidal Lipid Droplets in Chlamydomonas reinhardtii[END_REF]Moellering & Benning, 2010;[START_REF] Nguyen | Proteomic profiling of oil bodies isolated from the unicellular green microalga Chlamydomonas reinhardtii : With focus on proteins involved in lipid metabolism[END_REF][START_REF] Tsai | Dynamics of protein and polar lipid recruitment during lipid droplet assembly in Chlamydomonas reinhardtii[END_REF], as well as from secondary endosymbionts such as Phaeodactylum tricornutum (Leyland et al., 2020b;[START_REF] Lupette | The architecture of lipid droplets in the diatom Phaeodactylum tricornutum[END_REF] have

shown that their LDs contain high amounts of proteins involved in lipid synthesis. Most can be directly related to TAG biosynthesis (such as enzymes involved in FA synthesis, GPATs, LPATs and DGATs, see Figure 3), while others are involved in different lipid synthesis pathways. Interestingly, the DGTS synthase BTA1 has been identified in the LD proteomes of the green algae Dunaliella bardawil [START_REF] Davidi | Proteome analysis of cytoplasmatic and plastidic beta-carotene lipid droplets in Dunaliella bardawil[END_REF], Chromochloris zofingiensis (X. [START_REF] Wang | Proteomics Analysis of Lipid Droplets from the Oleaginous Alga Chromochloris zofingiensis Reveals Novel Proteins for Lipid Metabolism[END_REF] and Chlamydomonas reinhardtii, in which it is one of the most abundant proteins [START_REF] Goold | Saturating Light Induces Sustained Accumulation of Oil in Plastidal Lipid Droplets in Chlamydomonas reinhardtii[END_REF][START_REF] Nguyen | Proteomic profiling of oil bodies isolated from the unicellular green microalga Chlamydomonas reinhardtii : With focus on proteins involved in lipid metabolism[END_REF] C. H. [START_REF] Tsai | Dynamics of protein and polar lipid recruitment during lipid droplet assembly in Chlamydomonas reinhardtii[END_REF]. It has been proposed that LD-resident BTA1 synthetizes the DGTS that surrounds Chlamydomonas LDs [START_REF] Goold | Saturating Light Induces Sustained Accumulation of Oil in Plastidal Lipid Droplets in Chlamydomonas reinhardtii[END_REF][START_REF] Nguyen | Proteomic profiling of oil bodies isolated from the unicellular green microalga Chlamydomonas reinhardtii : With focus on proteins involved in lipid metabolism[END_REF]. However, while DGTA is found in the monolayer surrounding Phaeodactylum LDs [START_REF] Lupette | The architecture of lipid droplets in the diatom Phaeodactylum tricornutum[END_REF] the putative DGTA synthase has not been identified in the corresponding LD proteomes (Leyland et al., 2020b;[START_REF] Lupette | The architecture of lipid droplets in the diatom Phaeodactylum tricornutum[END_REF]. This could be explained by differences in experimentation time-courses, as LD proteomes from green algae were determined after 24-48h of nitrogen starvation while those of Phaeodactylum were established after 7 days. However, the different growth rates of these different algae make the comparison difficult. Alternatively, it could reflect metabolic differences between the considered organisms; DGTA is only a minor lipid in Phaeodactylum, while DGTS is major in Chlamydomonas where it replaces Phosphatidylcholine (PC).

Several proteins involved in sterol biosynthesis have also been identified in many LD proteomes. This is the case after nitrogen starvation in the previously cited green algae [START_REF] Davidi | Proteome analysis of cytoplasmatic and plastidic beta-carotene lipid droplets in Dunaliella bardawil[END_REF][START_REF] Goold | Saturating Light Induces Sustained Accumulation of Oil in Plastidal Lipid Droplets in Chlamydomonas reinhardtii[END_REF][START_REF] Nguyen | Proteomic profiling of oil bodies isolated from the unicellular green microalga Chlamydomonas reinhardtii : With focus on proteins involved in lipid metabolism[END_REF][START_REF] Tsai | Dynamics of protein and polar lipid recruitment during lipid droplet assembly in Chlamydomonas reinhardtii[END_REF]X. Wang et al., 2019) and in the diatom Phaeodactylum tricornutum (Leyland et al., 2020b), as well as in Chlamydomonas after exposure to high light. One of these proteins, the Sterol methyl-transferase 1, has also been identified in the proteome of Nicotiana benthamiana pollen tube LDs, along with cycloartenol synthase, another enzyme of sterol metabolism [START_REF] Kretzschmar | PUX10 Is a Lipid Droplet-Localized Scaffold Protein That Interacts with CELL DIVISION CYCLE48 and Is Involved in the Degradation of Lipid Droplet Proteins[END_REF]. Interestingly, a function of LDs in sterol metabolism has been shown in yeast [START_REF] Sorger | A Yeast Strain Lacking Lipid Particles Bears a Defect in Ergosterol Formation *[END_REF], but whether a similar function exists in algae or plants remains unknown.

Altogether, the presence of proteins involved in different lipid synthesis pathways in several independent LD proteomes raises questions as to potential roles of LDs in coordinating lipid production pathways in the considered organisms/organs.

In addition to proteins involved in lipid synthesis, proteins involved in membrane remodeling and lipid trafficking have also been found in the previously cited proteomes as well as in another diatom, Fistulifera solaris [START_REF] Nonoyama | Proteomics analysis of lipid droplets indicates involvement of membrane trafficking proteins in lipid droplet breakdown in the oleaginous diatom Fistulifera solaris[END_REF], along with cytoskeleton and intracellular vesicle trafficking proteins. Several studies have pointed out the function of LDs in lipid remodeling. In the absence of stress, TAG synthesis in Chlamydomonas reinhardtii is mainly dependent on PDAT. This enzyme (CrPDAT) can not only use phospholipids but also the plastid lipids MGDG, DGDG and SQDG as fatty acid donors (cf. Figure 2). Moreover, reduction of CrPDAT levels results in a slightly slower growth, suggesting that lipid remodeling by this pathway is important for the cell fitness [START_REF] Yoon | Phospholipid:Diacylglycerol Acyltransferase Is a Multifunctional Enzyme Involved in Membrane Lipid Turnover and Degradation While Synthesizing Triacylglycerol in the Unicellular Green Microalga Chlamydomonas reinhardtii[END_REF].

While this study shows that CrPDAT contribution is less important in stress conditions, degradation of glycerolipids is still important to feed TAGs under such situations. The disruption of the lipase PLASTID GALACTOGLYCEROLIPID DEGRADATION1 (PGD1), which hydrolyses MGDG, reduced TAG accumulation under nitrogen starvation (Li et al., 2012a), while other classes of glycerolipids are converted to TAGs under heat stress [START_REF] Légeret | Lipidomic and transcriptomic analyses of Chlamydomonas reinhardtii under heat stress unveil a direct route for the conversion of membrane lipids into storage lipids[END_REF]. This recycling of glycerolipids to TAGs is likely conserved as high levels of very long-chain unsaturated fatty acids are found in TAGs in Stramenopiles (reviewed in [START_REF] Guéguen | Lipid Droplets in Unicellular Photosynthetic Stramenopiles[END_REF]) as well as in the Haptophyte Tisochrysis lutea (B. [START_REF] Huang | Betaine lipid and neutral lipid production under nitrogen or phosphorus limitation in the marine microalga Tisochrysis lutea (Haptophyta)[END_REF] following nitrogen or phosphate deprivation. Finally, study of the green microalga Parachlorella kessleri under salt stress has shown that LDs accumulate at the cell periphery and likely contribute to remodeling the FA composition of the plasmid membrane lipids to modify its permeability [START_REF] You | Lipid Droplets Mediate Salt Stress Tolerance in Parachlorella kessleri[END_REF].

Lipid Droplets and lipid remodeling in the pollen tube

In higher plants, lipid trafficking appears as a major function during pollen formation and later during pollen tube growth. Sporopollenin, a very resistant polymer that covers pollen, is synthetized and deposited from a specific tissue localized in the anther and called the tapetum. Tapetum cells form specific LDs associated with ER membranes, named tapetosomes, as well as specialized plastids filled with plastoglobuli, called elaioplasts. Upon programmed cell death, tapetal cells release the lipids contained in those structures that contribute to the formation of the pollen coat (reviewed by [START_REF] Ischebeck | Lipids in pollen-They are different[END_REF]). Pollen germination and pollen tube growth require LD mobility. Indeed, in olive pollen after germination LD are no more found in the grain, but many are present in the pollen tube (K. [START_REF] Zienkiewicz | Identification and localization of a caleosin in olive (Olea europaea L.) pollen during in vitro germination[END_REF]. This localization change of LDs is hindered by the enlarged size of LDs in triple Seipin mutants of Arabidopsis, and leads to male fertility defects [START_REF] Taurino | SEIPIN Proteins Mediate Lipid Droplet Biogenesis to Promote Pollen Transmission and Reduce Seed Dormancy[END_REF]. The continuous TAG synthesis during pollen tube growth is at least partly fed by the recycling of phospholipids. Indeed, mutants deficient for the non-specific phospholipases C2 and C6 [START_REF] Bose | Non-specific phospholipases C2 and C6 redundantly function in pollen tube growth via triacylglycerol production in Arabidopsis[END_REF] as well as for DGAT1 and PDAT1 [START_REF] Bose | Non-specific phospholipases C2 and C6 redundantly function in pollen tube growth via triacylglycerol production in Arabidopsis[END_REF][START_REF] Zhang | DGAT1 and PDAT1 Acyltransferases Have Overlapping Functions in Arabidopsis Triacylglycerol Biosynthesis and Are Essential for Normal Pollen and Seed Development[END_REF] (cf. Figure 2) show reduced TAG synthesis. In his 2016 review, Ischebeck proposed several interesting hypotheses as to the function of LDs in pollen tube, including the involvement of LDs in lipid transport, regulation of lipid synthesis, acyl-editing or adaptation to environmental changes occurring during the pollen tube growth [START_REF] Ischebeck | Lipids in pollen-They are different[END_REF]. These functions have been known in other organisms for some time, in particular mammals [START_REF] Murphy | The dynamic roles of intracellular lipid droplets : From archaea to mammals[END_REF], and may not be restricted to the pollen tube.

Lipid Droplets and lipid remodeling in leaves

In higher plants, TAGs represent a very low percentage of glycerolipids in healthy leaves and very few LDs are observed. However, disruption of TAG degradation leads to ectopic accumulation of LDs in the leaves [START_REF] James | Disruption of the Arabidopsis CGI-58 homologue produces Chanarin-Dorfman-like lipid droplet accumulation in plants[END_REF], suggesting that TAG are constantly synthetized and degraded in this tissue. Later studies have shown that LD abundance varies with the diurnal cycle, and that LDs are the most abundant at the end of the night phase [START_REF] Gidda | Lipid Droplet-Associated Proteins (LDAPs) Are Required for the Dynamic Regulation of Neutral Lipid Compartmentation in Plant Cells[END_REF]. As FA synthesis is promoted by photosynthesis, it seems unlikely that these TAG are synthetized de novo and more likely that they derive from recycling of membrane lipids. Mechanisms explaining TAG function in lipid homeostasis and remodeling in higher plants have been proposed in a recent review [START_REF] Yu | Mechanisms and functions of membrane lipid remodeling in plants[END_REF]. Indeed, the Arabidopsis genome encodes three different DGATs and two PDATs with different substrate specificities in terms of acyl chain length and insaturation levels. Therefore, expression levels of the various DGATs may affect the acyl-CoA pool available for other lipid productions, while expression levels of the PDATs may promote recycling of certain PC species, thus modifying membrane compositions. As TAG storage organelles, LDs could just serve as a transitory sink or help to preserve certain fatty acids that are costly to produce or that can be used in case of rapidly changing conditions. Interestingly, a recent paper has enlightened the important role of autophagy for TAG formation and membrane lipid turnover in mature and senescent leaves, highlighting a possible role of LDs for the degradation of damaged organelles and recycling of their lipid contents [START_REF] Fan | Dual Role for Autophagy in Lipid Metabolism in Arabidopsis[END_REF].

Indeed, LD formation in leaves is mainly triggered by senescence and abiotic stresses. During senescence, the degradation of membrane lipids and subsequent formation of TAGs may allow the redistribution of FA to other tissues. Consistently, the proteome of LDs in senescent leaves contains several cytoskeleton proteins as well as proteins involved in lipid but also protein transports [START_REF] Brocard | Proteomic Analysis of Lipid Droplets from Arabidopsis Aging Leaves Brings New Insight into Their Biogenesis and Functions[END_REF]. In addition to the formation of LDs, abiotic stresses trigger changes in the fatty acid composition of glycerolipids, in particular of the plastid lipids MGDG and DGDG, and of PC. These changes vary depending on the type of stress, as does TAG composition (reviewed in [START_REF] Higashi | Lipidomic studies of membrane glycerolipids in plant leaves under heat stress[END_REF]). Heat, drought and salt stress can induce a very rapid TAG accumulation, in contrast to cold, high light or osmotic stress that require a longer exposure [START_REF] Mueller | Accumulation of extra-chloroplastic triacylglycerols in Arabidopsis seedlings during heat acclimation[END_REF]. The response to heat stress has been extensively studied and shows that TAG generation comes from the recycling of other lipids rather than from de novo synthesis [START_REF] Higashi | Lipidomic studies of membrane glycerolipids in plant leaves under heat stress[END_REF]. Indeed, transcriptomic data indicate a decrease of fatty acid and chloroplast glycerolipid syntheses [START_REF] Higashi | Landscape of the lipidome and transcriptome under heat stress in Arabidopsis thaliana[END_REF], while the generated TAGs contain high levels of unsaturated fatty acids, in particular C18:3 and C16:3 [START_REF] Higashi | Landscape of the lipidome and transcriptome under heat stress in Arabidopsis thaliana[END_REF][START_REF] Mueller | Accumulation of extra-chloroplastic triacylglycerols in Arabidopsis seedlings during heat acclimation[END_REF]. Analysis of fatty acid desaturation mutants shows that these unsaturated fatty acids are likely recycled from MGDG [START_REF] Mueller | Phospholipid:Diacylglycerol Acyltransferase-Mediated Triacylglyerol Synthesis Augments Basal Thermotolerance[END_REF], after the initial removal of C18:3 from MGDG by the Heat-Inducible Lipase 1 (HIL1) [START_REF] Higashi | HEAT INDUCIBLE LIPASE1 Remodels Chloroplastic Monogalactosyldiacylglycerol by Liberating α-Linolenic Acid in Arabidopsis Leaves under Heat Stress[END_REF]. The analysis of several TAG synthesis mutants has shown that PDAT1, but not DGAT1, is the major actor in TAG biosynthesis upon heat stress [START_REF] Mueller | Phospholipid:Diacylglycerol Acyltransferase-Mediated Triacylglyerol Synthesis Augments Basal Thermotolerance[END_REF]). Finally, the thermosensitivity observed in pdat1 and hil1 mutants enlightens the importance of MGDG remodeling and of TAG synthesis in this process [START_REF] Higashi | HEAT INDUCIBLE LIPASE1 Remodels Chloroplastic Monogalactosyldiacylglycerol by Liberating α-Linolenic Acid in Arabidopsis Leaves under Heat Stress[END_REF][START_REF] Mueller | Phospholipid:Diacylglycerol Acyltransferase-Mediated Triacylglyerol Synthesis Augments Basal Thermotolerance[END_REF]. MGDG remodeling also appears essential in tolerance to freezing, albeit for different reasons. Indeed, Moellering and collaborators have identified Sensitiveto-Freezing2 (SFR2) as a galactolipid:galactolipid galactosyltransferase that can transfer galactose from MGDG to another galactolipid, thus depleting the MGDG pool and forming oligogalactolipids and DAGs, the latter being converted into TAGs (cf. Figure 2). The authors hypothesized that this reduction of MGDG and consequent increase in DGDG and other oligogalactolipids serve to stabilize membranes following freezing-induced desiccation (Moellering et al., 2010). Consistently, similar changes in galactolipids and TAGs were observed upon drought [START_REF] Gasulla | The role of lipid metabolism in the acquisition of desiccation tolerance in Craterostigma plantagineum : A comparative approach[END_REF][START_REF] Tarazona | An enhanced plant lipidomics method based on multiplexed liquid chromatography-mass spectrometry reveals additional insights into coldand drought-induced membrane remodeling[END_REF] and cold [START_REF] Degenkolbe | Differential remodeling of the lipidome during cold acclimation in natural accessions of Arabidopsis thaliana[END_REF][START_REF] Tarazona | An enhanced plant lipidomics method based on multiplexed liquid chromatography-mass spectrometry reveals additional insights into coldand drought-induced membrane remodeling[END_REF] stresses, and SFR2 was also associated with resistance to salt and drought stresses in tomato (Wang et al., 2016). More recent studies have shown that in contrast to heat stress, DGAT1 is the essential player in TAG formation upon freezing and is important for freezing tolerance (Arisz et al., 2018;[START_REF] Tan | DIACYLGLYCEROL ACYLTRANSFERASE and DIACYLGLYCEROL KINASE Modulate Triacylglycerol and Phosphatidic Acid Production in the Plant Response to Freezing Stress[END_REF].

Lipid droplets and cell protection

In addition to their role in membrane lipid remodeling and transitory storage of fatty acids associated with stress conditions, leaf LDs appear essential to cell protection. Indeed, free fatty acids, as well as glycerolipids that cannot assemble into membranes such as PA or DAG, can be toxic for the cell by disorganizing the complex intracellular membrane network [START_REF] Li | Excess diacylglycerol at the endoplasmic reticulum disrupts endomembrane homeostasis and autophagy[END_REF][START_REF] Petschnigg | Good Fat, Essential Cellular Requirements for Triacylglycerol Synthesis to Maintain Membrane Homeostasis in Yeast[END_REF].

Storage into TAG serves as a transient but essential sink before degradation [START_REF] Fan | Arabidopsis Lipins, PDAT1 Acyltransferase, and SDP1 Triacylglycerol Lipase Synergistically Direct Fatty Acids toward β-Oxidation, Thereby Maintaining Membrane Lipid Homeostasis[END_REF][START_REF] Fan | A Central Role for Triacylglycerol in Membrane Lipid Breakdown, Fatty Acid β-Oxidation, and Plant Survival under Extended Darkness[END_REF].

In addition to TAG and sterol esters, LDs can store other hydrophobic compounds such as carotenoids. In the marine green algae Dunaliella bardawil and salina, carotenoids are stored in plastoglobuli, while in freshwater green algae such as Haematococcus pluvialis, carotenoids are found in cytosolic LDs. In both cases, carotenoid production is induced by reactive oxygen species (ROS) that are generated following stress such as high light or nutrient deprivation, and they can serve both to protect the photosynthetic system against photodamage created by excessive irradiation and to quench the effect of ROS (review [START_REF] Pick | A hypothesis about the origin of carotenoid lipid droplets in the green algae Dunaliella and Haematococcus[END_REF]). Carotenoids have also been found in Phaeodactylum LDs but whether they are stored in cytosolic LDs or plastoglobuli is not clear [START_REF] Lupette | The architecture of lipid droplets in the diatom Phaeodactylum tricornutum[END_REF].

Finally, several proteins of the ER associated protein degradation (ERAD) pathway, which serve to remove misfolded proteins and target them for degradation by the 26S-proteasome, have been identified in LD proteomes of the diatom Phaeodactylum tricornutum after 7 days of nitrogen deprivation (Leyland et al., 2020b;[START_REF] Lupette | The architecture of lipid droplets in the diatom Phaeodactylum tricornutum[END_REF] as well as in Arabidopsis seeds [START_REF] Deruyffelaere | PUX10 Is a CDC48A Adaptor Protein That Regulates the Extraction of Ubiquitinated Oleosins from Seed Lipid Droplets in Arabidopsis[END_REF][START_REF] Kretzschmar | PUX10 Is a Lipid Droplet-Localized Scaffold Protein That Interacts with CELL DIVISION CYCLE48 and Is Involved in the Degradation of Lipid Droplet Proteins[END_REF], senescent leaves [START_REF] Fernandez-Santos | Protein Profiles of Lipid Droplets during the Hypersensitive Defense Response of Arabidopsis against Pseudomonas Infection[END_REF] and Nicotiana pollen [START_REF] Kretzschmar | PUX10 Is a Lipid Droplet-Localized Scaffold Protein That Interacts with CELL DIVISION CYCLE48 and Is Involved in the Degradation of Lipid Droplet Proteins[END_REF]. Their function in Arabidopsis seeds seems to be mainly the degradation of Oleosins and Caleosins preceding the remobilization of oil reserves upon germination [START_REF] Deruyffelaere | Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis[END_REF][START_REF] Deruyffelaere | PUX10 Is a CDC48A Adaptor Protein That Regulates the Extraction of Ubiquitinated Oleosins from Seed Lipid Droplets in Arabidopsis[END_REF]. However, many additional proteins associated with the regulation of protein folding and ubiquitination have been identified in LDs from pollen tubes, leaves and Phaeodactylum, suggesting a broader function of lipid droplets in protein processing and catabolism.

Lipid droplets, a reserve to restart

In addition to proteins associated with lipid metabolism or LD structure that are expected to be present in LDs, much more unexpected proteins are found. In many cases, they are assumed to be the result of contaminations, and, as such, are little discussed in the literature. Yet, there are lines of evidence suggesting that they should be considered with more attention.

Many of the microalgae LD proteomes performed under nutrient starvation conditions include proteins linked to DNA organization (histones), and regulation of protein translation (ribosomal proteins, translation elongation factors) [START_REF] Davidi | Proteome analysis of cytoplasmatic and plastidic beta-carotene lipid droplets in Dunaliella bardawil[END_REF][START_REF] Goold | Saturating Light Induces Sustained Accumulation of Oil in Plastidal Lipid Droplets in Chlamydomonas reinhardtii[END_REF]Leyland et al., 2020b;[START_REF] Lupette | The architecture of lipid droplets in the diatom Phaeodactylum tricornutum[END_REF]Moellering & Benning, 2010;[START_REF] Nguyen | Proteomic profiling of oil bodies isolated from the unicellular green microalga Chlamydomonas reinhardtii : With focus on proteins involved in lipid metabolism[END_REF][START_REF] Tsai | Dynamics of protein and polar lipid recruitment during lipid droplet assembly in Chlamydomonas reinhardtii[END_REF][START_REF] You | Lipid Droplets Mediate Salt Stress Tolerance in Parachlorella kessleri[END_REF]. Interestingly, these are generally not found in higher plant seed proteomes.

Upon nutrient starvation, LDs can include all histones or a subset of histones, including core histones (H2A, H2B, H3 and H4), histone variants, as well as the linker histone H1. While histones are abundant proteins, they are mostly contained in the nucleus and are highly hydrophilic proteins, suggesting that their association with LDs in stressed microalgae is unlikely to result from contamination. The association of histones to LDs has been described in the model fly Drosophila melanogaster, where LDs serve as histone storage [START_REF] Cermelli | The Lipid-Droplet Proteome Reveals that Droplets Are a Protein-Storage Depot[END_REF] and regulate histone supply during the early phases of embryonic development (Li et al., 2012b). As nutrient starvation severely impairs microalgae growth, it could be speculated that LDs, similarly to what occurs in Drosophila, could serve as a histone deposit during stress, when cell growth is severely impaired, to favor quick restart once the conditions become more favorable. The storage of ribosomal proteins and translation elongation factors could serve the same purpose. Ribosomal proteins are among the most abundant proteins in the cell and their presence in LD proteomes has long been considered as contaminations. Yet, similarly to histones, they are not found in plant seeds proteomes. Moreover, proteins associated with translation processes are the most strongly down-regulated upon nitrogen and phosphorus starvation [START_REF] Hulatt | Proteomic and Transcriptomic Patterns during Lipid Remodeling in Nannochloropsis gaditana[END_REF], and storage in LDs could serve to preserve a minimal pool from degradation. In support of this possible role for LDs, neither histones nor translationassociated proteins are found in the LD proteome of Chlamydomonas grown under high light, a condition that triggers LD accumulation but not cell growth arrest [START_REF] Goold | Saturating Light Induces Sustained Accumulation of Oil in Plastidal Lipid Droplets in Chlamydomonas reinhardtii[END_REF].

Finally, some RNA-binding proteins have been identified in several microalgae proteomes [START_REF] Lupette | The architecture of lipid droplets in the diatom Phaeodactylum tricornutum[END_REF]Moellering & Benning, 2010;[START_REF] Nguyen | Proteomic profiling of oil bodies isolated from the unicellular green microalga Chlamydomonas reinhardtii : With focus on proteins involved in lipid metabolism[END_REF]X. Wang et al., 2019), raising the possibility that LDs could serve to preserve a RNA pool.

Lipid droplets and brassinosteroid signaling

Together with Oleosins and Caleosins, Steroleosins are one of the three families of proteins associated with LDs in plants. They may be associated with the synthesis of brassinosteroids, which are plant hormones involved in different developmental processes as well as resistance to biotic and abiotic stresses [START_REF] Divi | Brassinosteroid : A biotechnological target for enhancing crop yield and stress tolerance[END_REF][START_REF] Singh | Growth control : Brassinosteroid activity gets context[END_REF]. The first two steroleosins have been identified in Sesamum indicum [START_REF] Chen | Identification of Three Novel Unique Proteins in Seed Oil Bodies of Sesame[END_REF], and the first identified Arabidopsis homolog possesses 11and-hydroxysteroid dehydrogenase activities and was thus renamed HYDROXYSTEROID DEHYDROGENASE 1 (AtHSD1) (d'Andréa et al., 2007). Initial studies have shown that overexpression of AtHSD1 leads to reduced seed dormancy [START_REF] Baud | Regulation of HSD1 in Seeds of Arabidopsis thaliana[END_REF][START_REF] Li | A Putative Hydroxysteroid Dehydrogenase Involved in Regulating Plant Growth and Development[END_REF] as well as to increased growth and resistance to salt stress (F. [START_REF] Li | A Putative Hydroxysteroid Dehydrogenase Involved in Regulating Plant Growth and Development[END_REF]. How the association of AtHSD1 to LDs modulates its function and contributes to brassinosteroid signaling remain open questions.

LD and biotic stress

Interactions between LDs and pathogens have been mostly studied in mammalian cells and have highlighted two antagonist roles for LDs, which can either be used by the pathogen or participate in cell and organism defense.

Hijacking of the host metabolism

The current state of the art suggests that mammalian LDs can participate in the infection process, providing invaders with substrates for survival and/or growth [START_REF] Bosch | Mammalian lipid droplets are innate immune hubs integrating cell metabolism and host defense[END_REF]. This phenomenon is described as the "hijacking" of the host metabolism. Bacteria such as Chlamydia trachomatis [START_REF] Kumar | The Obligate Intracellular Pathogen Chlamydia trachomatis Targets Host Lipid Droplets[END_REF], eukaryotic parasites such as Toxoplasma gondii and Neospora caninum [START_REF] Hu | The coccidian parasites Toxoplasma and Neospora dysregulate mammalian lipid droplet biogenesis[END_REF], or even viruses such as Hepatitis C Virus [START_REF] Herker | Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1[END_REF][START_REF] Miyanari | The lipid droplet is an important organelle for hepatitis C virus production[END_REF] and Sars-Cov2 [START_REF] Dias | Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators[END_REF], can induce the accumulation of LDs in the host and use it as a lipid source. During viral infection, this hijacking can go even further. For example, the Hepatitis C Virus (HCV) targets the liver, where LDs are consumed for the production and assembly of new infectious virus particles. In a model proposed by Miyanari et al, the core HCV protein binds to the lipid monolayer surrounding the LD, brings ER membranes close to the LD and recruits virus proteins as well as replication complexes to this microenvironment in order to produce new viral particles [START_REF] Miyanari | The lipid droplet is an important organelle for hepatitis C virus production[END_REF].

Interestingly, this hijacking of the host metabolism by pathogens also occurs in viral infection of eukaryotic microalgae. Due to high ecological relevance, several studies have taken interest in the infection of Haptophytes, and in particular of the coccolithophore Emiliana Huxleyi, by large dsDNA

viruses. An initial study showed that infection of Emiliana by the lytic virus EhV201, belonging to the Phycodnaviridae, leads to a complete rewiring of the host lipidome [START_REF] Rosenwasser | Rewiring Host Lipid Metabolism by Large Viruses Determines the Fate of Emiliania huxleyi, a Bloom-Forming Alga in the Ocean[END_REF]). An increase in de novo fatty acid synthesis proved crucial for viral production. Indeed, lipid biosynthesis reoriented to favor the production of specific glycosphingolipids at the expense of host sphingolipids.

These virus-derived glycosphingolipids (vGSL) are central structural components of the virus membrane and can trigger programmed cell death of infected cell [START_REF] Vardi | Viral Glycosphingolipids Induce Lytic Infection and Cell Death in Marine Phytoplankton[END_REF]. Finally, sterols appeared depleted in the host, feeding the new virions [START_REF] Rosenwasser | Rewiring Host Lipid Metabolism by Large Viruses Determines the Fate of Emiliania huxleyi, a Bloom-Forming Alga in the Ocean[END_REF]. [START_REF] Malitsky | Viral infection of the marine alga Emiliania huxleyi triggers lipidome remodeling and induces the production of highly saturated triacylglycerol[END_REF] completed this study and showed that Ehv201 can induce specific TAG accumulation and LD formation by upregulating genes involved in de novo TAG synthesis (LPAT, PAP, DGAT2) and downregulating genes involved in TAG recycling from phospholipids (such as PDAT). Virus-induced TAG, characterized by a high enrichment in unsaturated and monounsaturated fatty acids, are later found in high amounts in the virions. All those results led the authors to speculate that virus-induced LDs play a crucial role in virus assembly [START_REF] Malitsky | Viral infection of the marine alga Emiliania huxleyi triggers lipidome remodeling and induces the production of highly saturated triacylglycerol[END_REF] (Figure 4, left panel) as has been described for HCV [START_REF] Miyanari | The lipid droplet is an important organelle for hepatitis C virus production[END_REF]. Other virus families may share this mechanism. Indeed, a recent study showed that the giant haptophyte-infecting virus, Prymnesium kappa virus RF01 (PkV RF01) belonging to the Mimiviridae family, encodes for many proteins involved in lipid catabolism, among which a TAG lipase and other key enzymes of the -oxidation pathway. This suggests that this virus is also capable to break down host LDs for its propagation [START_REF] Blanc-Mathieu | A Persistent Giant Algal Virus, with a Unique Morphology, Encodes an Unprecedented Number of Genes Involved in Energy Metabolism[END_REF].

Lipid Droplets as defense organelles

While this LD hijacking by pathogens may paint LDs as weak points in the cell, their active role in innate defense have been equally reviewed [START_REF] Pereira-Dutra | Fat, fight, and beyond : The multiple roles of lipid droplets in infections and inflammation[END_REF]. Indeed, in mammals, proteins with anti-pathogen properties such as viperin [START_REF] Hinson | The antiviral protein, viperin, localizes to lipid droplets via its Nterminal amphipathic α-helix[END_REF] and interferon- (IFN-) inducible guanosine triphosphatase (GTPase) (IGTP [START_REF] Bougnères | A Role for Lipid Bodies in the Cross-presentation of Phagocytosed Antigens by MHC Class I in Dendritic Cells[END_REF][START_REF] Haldar | IRG and GBP Host Resistance Factors Target Aberrant, "Non-self" Vacuoles Characterized by the Missing of "Self" IRGM Proteins[END_REF]) are localized in the LDs of infected cells, and similar roles of LDs have been shown in higher plants.

Several studies regarding LDs in Arabidopsis thaliana leaves highlight their function in defense against various pathogens [START_REF] Brocard | Proteomic Analysis of Lipid Droplets from Arabidopsis Aging Leaves Brings New Insight into Their Biogenesis and Functions[END_REF][START_REF] Fernandez-Santos | Protein Profiles of Lipid Droplets during the Hypersensitive Defense Response of Arabidopsis against Pseudomonas Infection[END_REF][START_REF] Shimada | Leaf Oil Body Functions as a Subcellular Factory for the Production of a Phytoalexin in Arabidopsis[END_REF][START_REF] Shimada | Membrane Dynamics and Multiple Functions of Oil Bodies in Seeds and Leaves[END_REF]. Indeed, proteomics have shown that several enzymes involved in production of phytoalexins, compounds that participate in plant defense, are located in LDs. The most prominent example is that of Caleosins (CLO), which are structural components of plant LDs and exhibit a peroxygenase activity. CLO3, which is also known as Responsive to Dehydratation20 (RD20) can be induced by abiotic stresses, notably drought [START_REF] Aubert | RD20, a Stress-Inducible Caleosin, Participates in Stomatal Control, Transpiration and Drought Tolerance in Arabidopsis thaliana[END_REF] or salt, but also in response to the pathogenic fungus Colletotrichum higginsianum [START_REF] Shimada | Leaf Oil Body Functions as a Subcellular Factory for the Production of a Phytoalexin in Arabidopsis[END_REF]. Upon infection, the authors have shown that CLO3 colocalizes with α-Dioxygenase 1 (αDOX1) on LDs of perilesional cells of the infected leaf, where they coordinately use α-linolenic acid (C18:3, ω-3) to produce the antifungal oxylipin 2-hydroxy-octadecatrienoic acid (2-HOT). A similar effect is triggered by senescence and the authors propose that phytoalexin-producing LDs that develop in dying cells of perilesional tissues or senescent leaves could serve to form a shield in order to protect the young healthy parts of the plant from infection [START_REF] Shimada | Leaf Oil Body Functions as a Subcellular Factory for the Production of a Phytoalexin in Arabidopsis[END_REF] (Figure 4, right panel). A recent proteomics analysis comparing LDs from leaves infected by the bacterium Pseudomonas syringae pv.

Tomato (Pst) with senescent leaves has shown that they both contain enzymes involved in the biosynthesis of the antimicrobial compound camalexin, as well as GPAT4 and GPAT8, which are implicated in cutin biosynthesis [START_REF] Fernandez-Santos | Protein Profiles of Lipid Droplets during the Hypersensitive Defense Response of Arabidopsis against Pseudomonas Infection[END_REF]. The overlap between LD proteomes in these two conditions confirms that they likely share common functions in cell defense against pathogens. Finally, the ectopic expression of stilbene synthases from Vitis pseudoreticulata (VpSTS29) in Arabidopsis shows that these enzymes, which play a critical role in powdery mildew resistance, can relocate from the cytoplasm to cytosolic LDs upon dark-induced senescence [START_REF] Ma | Ectopic expression of VpSTS29, a stilbene synthase gene from Vitis pseudoreticulata, indicates STS presence in cytosolic oil bodies[END_REF]. Intriguingly, stilbene synthase-containing LDs can be found both in the cytoplasm and in the vacuole, where stilbenes seem to be stored, thus implying a trafficking role for LDs, an hypothesis supported by the association of many cytoskeleton proteins with LDs [START_REF] Brocard | Proteomic Analysis of Lipid Droplets from Arabidopsis Aging Leaves Brings New Insight into Their Biogenesis and Functions[END_REF][START_REF] Fernandez-Santos | Protein Profiles of Lipid Droplets during the Hypersensitive Defense Response of Arabidopsis against Pseudomonas Infection[END_REF]. Finally, AtCPK1, which is a Ca2+ dependent protein kinase involved in the response to fungal pathogens through the salicylic acid-mediated defense pathway, shows a dual localization to peroxisomes and LDs [START_REF] Coca | AtCPK1 calcium-dependent protein kinase mediates pathogen resistance in Arabidopsis[END_REF]. However, the function of this localization remains unknown.

Altogether, these results suggest that LDs of higher plants can be seen as production platforms for anti-pathogenic compounds involved in various plant defense mechanisms. However, we could not find in the literature any evidence that LDs play such a role in microalgae. The only potential link identified thus far lies in the antibacterial function of Eicosapentaenoic acid (EPA), a very long chain polyunsaturated fatty acid (VLC-PUFA) that is very abundant in many microalgae [START_REF] Desbois | A Fatty Acid from the Diatom Phaeodactylum tricornutum is Antibacterial Against Diverse Bacteria Including Multi-resistant Staphylococcus aureus (MRSA)[END_REF]. However, VLC-PUFA are generally not very abundant in TAGs, and potential anti-pathogenic activities of LDs remain to be investigated in these organisms. This difference of functions, if it exists, may be linked with the multicellular nature of higher plants where an infected part of the plant can signal the danger to the rest of the organism and trigger defenses. It would be therefore very interesting to investigate whether similar signals could exist at the population scale in microalgae.

Conversely, the hijacking of LDs has not yet been evidenced in plants even though it exists in multicellular organisms such as mammals. The function of LDs in defense against pathogens in plants and algae thus retains many mysteries to unravel.

Conclusion

In the present review, we have explored the many roles of LDs, far from the simple fat stock that they initially represented (Figure 5). It is not clear whether the same LDs carry all functions or if a cell can contain different types of LDs. Several lines of evidence suggest the latter. Analysis of LDs subpopulation in Phaeodactylum during nitrogen stress has shown that there are three waves of LD formation and that they exhibit different behaviours [START_REF] Jaussaud | Stepwise Biogenesis of Subpopulations of Lipid Droplets in Nitrogen Starved Phaeodactylum tricornutum Cells[END_REF]. The coexistence of several LDs population with distinct function has also been suggested from the analysis of Arabidopsis aging leaves [START_REF] Brocard | Proteomic Analysis of Lipid Droplets from Arabidopsis Aging Leaves Brings New Insight into Their Biogenesis and Functions[END_REF]. Finally, a refined analysis of LD at different stages of seed formation, germination and seedling development has shown many changes in their proteome, which may reflect production of different subsets of LDs [START_REF] Kretzschmar | Identification of Low-Abundance Lipid Droplet Proteins in Seeds and Seedlings[END_REF]. Our understanding of LD biogenesis as well as of their functions comes mostly from the study of non photosynthetic organisms, animals and yeast. Yet, while there are many common points, evidences

show that this knowledge cannot be simply extrapolated to photosynthetic organisms. The primary characteristic of plants and algae is the presence of a photosynthetic plastid that is central in the overall metabolism and in particular in the metabolism of lipids. Moreover, the evolutionary origin of this plastid can considerably diverge when we start looking at secondary endosymbionts, and its role in LD biogenesis requires further investigations. The study of LD roles enlightens the specificities of the responses to abiotic stresses in one given organism, as well as the differences that can exist between a single cell organism or a pluricellular one, between a primary or secondary endosymbiont, or even between freshwater algae or marine ones (Figure 5). Oleaginous microalgae are of great biotechnological interest, for the production of biofuels or high value-added molecules such as 3fatty acids or pigments, like EPA and astaxanthin, respectively. This has considerably increased the interest for these organisms. Unfortunately, many studies solely aim to increase the productivity of compounds of interest, without looking further into the biological or ecological relevance of the results. Therefore, knowledge on many species remains sparse and few studies compare the effects of different stresses. Yet, increasing our knowledge of LD functions and of their potential adaptive roles would be of great interest in the context of global change we are facing. The combination of genomics, proteomics, lipidomics and microscopy tools that are increasingly powerful provide exciting opportunities for future research. The LD core is mainly composed of TAGs with very diverse fatty acid compositions, and steryl esters.

Figures and

Other hydrophobic compounds can be found such as wax esters in certain land plants, and carotenoids and free fatty acids in some algae. Different classes of proteins can be found at the surface (A) and LD major proteins differ between organisms/tissues (B and C).

Figure 1 :

 1 Figure 1: Photosynthetic organisms in the tree of Life

Figure 2 :

 2 Figure 2: Structure and content of LDs

Acknowledgements:

The authors wish to thank Juliette Jouhet, Fabrice Rébeillé, Eric Maréchal and Alberto Amato for helpful discussion.

A: The different classes of proteins associated to the LD. Class I LD proteins are initially inserted via an hydrophobic hairpin structure in the ER membrane and are later transferred to the LD. Class II proteins are present in the cytosol and can become associated with the LDs. This association can be mediated by an amphipathic helix, or indirect, through protein-protein interaction or addition of a lipid anchor by post-translational modifications (adapted from [START_REF] Dhiman | Mechanisms of protein targeting to lipid droplets : A unified cell biological and biophysical perspective[END_REF]). Hydrophobic regions that mediate interaction with the droplet are indicated in red.

B: Major LD proteins in Stramenopiles. Predictions of major lipid droplet proteins in stramenopiles suggest that StLDP (Phaeodactylum tricornutum) and LDSP (Nannochloropsis s.l.) are class I proteins, with two hydrophobic -helixes forming a hairpin. On the other hand, DOAP1 (Fistulifera solaris) and PtLDP1 (Phaeodactylum tricornutum) are class II proteins; they do not contain a clear hydrophobic region but the central region is predicted to form an amphipathic -barrel (adapted from [START_REF] Guéguen | Lipid Droplets in Unicellular Photosynthetic Stramenopiles[END_REF]).

C: Major LD proteins in plants. Oleosins are the major and the most abundant proteins of seeds and pollen LD, while Caleosins and Steroleosins are less abundant but found in all plant tissues. Oleosins and Caleosins are class I proteins, strongly anchored in the LD through several hydrophobic β-sheets that form hairpin structures. They also contain a central "Proline knot" that penetrates into the LD core and is essential for targeting the Oleosins to the LD. Steroleosins are anchored to the LD by a "Proline knob" that mainly associates with the LD surrounding monolayer (adapted from [START_REF] Shao | New Insights Into the Role of Seed Oil Body Proteins in Metabolism and Plant Development[END_REF]). Plant leaves LDs are mainly covered by LDAPs and LDIP. LDIP is a class I protein with four transmembrane domains, while LDAPs are class II proteins that associate to the LD surface through their interactions with LDIP (adapted from [START_REF] Chapman | Mechanisms of lipid droplet biogenesis[END_REF]). Right panel: LD as a production platform for phytoalexins (based on [START_REF] Shimada | Leaf Oil Body Functions as a Subcellular Factory for the Production of a Phytoalexin in Arabidopsis[END_REF]. Response of Arabidopsis thaliana leaves to fungi infection. Upon infection by fungi such as Colletotrichum higginsianum, the expression of Caleosin3 (CLO3) and α-Dioxygenase 1 (αDOX1) is induced in perilesional cells, possibly through the action of abscissic acid (ABA), an essential hormone involved in the plant immune response (A). α-DOX1 and CLO3 locate to the leaf LD and cooperatively produce the oxylipin 2-hydroxyoctadecatrienoic acid (2-HOT) from α-linolenic acid (C18:3, ω-3) (B). 2-HOT blocks the fungi (F) spore germination and its propagation to healthy tissues (C). A similar defense system is induced by senescence.