Diana Molino

Jean-Denis Faure

Chapter SPHINGOLIPID VARIETY, BIOSYNTHESIS AND REGULATION

Sphingolipids constitute a ubiquitous class of lipids present in all eukaryotes as well as in several prokaryotes and even some viruses. Their structure is peculiar among other classes of lipids with a backbone made of a sphingoid base bearing an N-linked fatty acid. This structure, known as ceramide, is further modified by means of polar head substitutions. Head substitutions, fatty acids of different lengths and sphingoid bases variants generate a great assortment of sphingolipid isoforms (sphingolipidome), which is highly diversified between organisms, kingdoms and cell types. Sphingolipids are essential for organism development and cell growth since complex sphingolipids are major components of biological membranes and involved in membrane domain organization and receptor signaling. Sphingoid bases are instead involved in cell signaling, cell growth and stress responses. Several key enzymes of the biosynthetic pathway are highly conserved and finely regulated to generate key "structural orthologs" that seem to be shared among organisms. In this chapter, we describe step by step the sphingolipid biosynthetic pathway, and via a continuous parallel between plant, yeast and animal, we will review the sphingolipid functions in different organisms and cells.

INTRODUCTION

The assortment of sphingolipids (sphingolipidome) is highly diversified between organisms, kingdoms and cell types; however, several key enzymes of the biosynthetic pathway are highly conserved and several "structural orthologs" seem to be shared among organisms (Figure 1). The sphingolipid family gathers ceramides and complex sphingolipids derived from ceramide by addition of a variety of head substitutions on the first carbon (C1). For example, in the model plant Arabidopsis, the substitution of glucose or glucosylinositolphosphate leads to the formation of glucosyl-ceramide (GluCer) and glucosyl-inositolphosphoryl-ceramide (GIPC), respectively. GluCer and GIPC represent around 40 and 50% respectively of the sphingolipidome of Arabidopsis seedlings [START_REF] Markham | Plant sphingolipids: Separation and identification of major sphingolipid classes from leaves[END_REF]. GluCer is absent in Saccharomyces cervisiae, which only produces Inositolphosphorylceramide (IPC) and Mannosylated Inositolphosphoryl ceramide (MIPC) [START_REF] Lester | High-performance liquid chromatography analysis of molecular species of sphingolipid-related long chain bases and long chain base phosphates in Saccharomyces cerevisiae after derivatization with 6-aminoquinolyl-Nhydroxysuccinimidyl carbamate[END_REF]. However, homology search using BLAST revealed that several other fungi species contain GluCer synthase enzymes. In animal cells, GluCer is also synthetized, but rapidly converted in more complex glycosphingolipids.

Sphingomyelin (SM) is another abundant sphingolipid found only in animal cell membranes. SM is characterized by the presence of phosphocholine as head group, instead of a carbohydrate molecule like in other sphingolipids. SM is greatly enriched in the multilayered myelin sheath surrounding the axons of neurons, where it participates in the propagation of the electrical impulses. It has been proposed that the intertwining hydrocarbon chains of SM strengthen myelin sheath packing.

Besides different head substitution, variability is also observed in FA chain length and LCB and/or FA saturation and hydroxylation, leading to a multiplicity of sphingolipid isoforms. In particular, hydroxylation of FA and LCB moieties is a hallmark of sphingolipids. Sphingolipids are the lipid with the highest level of hydroxylation in plants. Sphingolipidome, with more than 160 different molecules, appears to be more diversified in plants compared to animals and yeast [START_REF] Markham | Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry[END_REF].

SPHINGOLIPID BIOSYNTHESIS

The Biosynthesis of the Fatty Acyl Chain

Sphingolipids, as many other lipids, contain a fatty acyl chain. Fatty acids (FA) of 16 or 18 carbon atoms (C16, C18) are synthetized by the fatty acid synthase complex and released for further use in the form of Acyl-CoAs. By definition, fatty acids of C16 or C18 are named LCFA for long chain fatty acid. The fatty acid synthase complex is localized in plant chloroplasts or animal mitochondria. LCFA-CoA can be further elongated to VLCFA-CoA (very long chain fatty acids) in the ER by the elongase complex. The elongase complex is composed of 4 enzymes that act in a sequential mode to add 2 carbons to the LCFA-CoA. Multiple cycles lead to VLCFA-CoA from 20C up to 26C [START_REF] Oh | ELO2 and ELO3, homologues of the Saccharomyces cerevisiae ELO1 gene, function in fatty acid elongation and are required for sphingolipid formation[END_REF][START_REF] Gaigg | Depletion of acyl-coenzyme A-binding protein affects sphingolipid synthesis and causes vesicle accumulation and membrane defects in Saccharomyces cerevisiae[END_REF] [START_REF] Han | The Saccharomyces cerevisiae YBR159w gene encodes the 3-ketoreductase of the microsomal fatty acid elongase[END_REF]. In Arabidopsis these enzymes are: 3-keto acyl-CoA synthase (KCS) [START_REF] Joubes | The VLCFA elongase gene family in Arabidopsis thaliana: phylogenetic analysis, 3D modelling and expression profiling[END_REF], 3-keto acyl-CoA reductase (KCR) [START_REF] Beaudoin | Functional characterization of the Arabidopsis betaketoacyl-coenzyme A reductase candidates of the fatty acid elongase[END_REF], 3-hydroxy acyl-CoA dehydratase (HCD/PASTICCINO2) [START_REF] Bach | The plant very long chain hydroxy fatty Acyl-CoA dehydratase PASTICCINO2 is essential and limiting for plant development[END_REF], and the enoyl-CoA reductase (ECR/CER10) [START_REF] Zheng | Disruptions of the Arabidopsis Enoyl-CoA reductase gene reveal an essential role for very-long-chain fatty acid synthesis in cell expansion during plant morphogenesis[END_REF]. VLCFA-CoA can then be incorporated in membrane lipids (sphingolipids, phospholipids); in storage lipids (triacylglycerides) or extracellular protective lipid (waxes and suberin). The length of the FA chain is a critical feature of sphingolipids. In both animals and plants, it can be long (LCFA: C16-18), or very long (VLCFA: C20-26), while in yeast, sphingolipids only contain VLCFA (C24-26). Sphingolipid biosynthesis is therefore tightly associated with FA elongation in yeast, while in plants and animals, two branched pathways exist, one linked to FA elongation, and the other not. In both animals and plants, LCFA-C18 and VLCFA-C24 are both significantly abundant and they may fulfill different functions. Phospholipids may also contain VLCFA but the great majority of them contain two symmetric C16/C18 chains. Finally, most of the FAs associated with sphingolipids are hydroxylated and saturated or mono-unsaturated.

In summary sphingolipids are characterized by very long, saturated or monounsaturated and hydroxylated acyl chains.

Sphingoid Bases Variants: Hydroxylation Is Essential in Plants while Saturation Predominates in Animals

The first step of the biosynthesis of the sphingoid backbone is the condensation of a serine with palmitoyl-CoA, via a serine palmitoyl transferase (SPT) (Table 1, Figure 2). The product is a long chain base called 3-ketosphinganin, which is immediately reduced by the ketosphinganin reductase (KSR) to sphinganine (Table 1). Genes encoding SPT are present in a large number of species from bacteria to humans and highly conserved among eukaryotes [START_REF] Sperling | Plant sphingolipids: structural diversity, biosynthesis, first genes and functions[END_REF][START_REF] Lynch | An introduction to plant sphingolipids and a review of recent advances in understanding their metabolism and function[END_REF]. The importance of sphingolipids in cell biology is highlighted by the deleterious consequence of the complete knockout of SPT. Mutations in SPT subunits have been involved in hereditary neuropathy in humans [START_REF] Dawkins | Mutations in SPTLC1, encoding serine palmitoyltransferase, long chain base subunit-1, cause hereditary sensory neuropathy type I[END_REF]. The complete knockout of SPT resulted in lethality in mice, yeast and plants [START_REF] Hojjati | Serine palmitoyl-CoA transferase (SPT) deficiency and sphingolipid levels in mice[END_REF][START_REF] Chen | The Essential Nature of Sphingolipids in Plants as Revealed by the Functional Identification and Characterization of the Arabidopsis LCB1 Subunit of Serine Palmitoyltransferase[END_REF] revealing that LCBs are essential molecules for cell viability. SPT functions as a heterodimer, and a 20% reduction of its activity by the inactivation of one subunit is sufficient for impairing cell expansion in Arabidopsis [START_REF] Chen | The Essential Nature of Sphingolipids in Plants as Revealed by the Functional Identification and Characterization of the Arabidopsis LCB1 Subunit of Serine Palmitoyltransferase[END_REF]. A complete list of all enzymes involved in the biosynthesis and modifications of sphingolipids in Arabidopsis and their subcellular localization is listed in Table 1. The LCB moiety always contains 18 carbon atoms chain that can be hydroxylated and desaturated. Such chemical modifications add structural variability to sphingolipids. A total extract of LCBs from Arabidopsis reveals a majority of di-and trihydroxylated LCBs (d18 or t18). Di-hydroxylation is associated with the presence of hydroxyl groups on C1 and C3, corresponding to the serine and palmitoyl-CoA precursors, respectively, while the third hydroxylation occurs later in the pathway on C4 by a C4hydroxylase (Figure 2). In Arabidopsis, two redundant genes, SBH1 and SBH2 (for sphingoid base hydroxylase 1 and 2, Table 1), encode a C4-hydroxylase. Double mutants as well as RNAi suppression lines revealed the physiological and metabolic relevance of this hydroxyl group [START_REF] Chen | Sphingolipid long-chain base hydroxylation is important for growth and regulation of sphingolipid content and composition in Arabidopsis[END_REF]. The complete loss of t18 (tri-hydroxyl LCB) resulted in both cell division and cell expansion defects, stressing the importance of LCB hydroxylation in sphingolipid cellular function. Moreover, the total content of sphingolipids in sbh mutants was globally modified with higher levels of d18 (di-hydroxyl LCB), as predicted. Unexpectedly, these mutants also showed higher levels of sphingolipids with shorter fatty acyl chains. These observations suggest that the 3-hydroxyl configuration (t18) is preferentially combined with VLCFA, while d18 is mainly associated with LCFA. Chen at al. proposed that the presence of tri-hydroxyl groups would be an important structural determinant for channelling VLCFA downstream in complex sphingolipids [START_REF] Chen | Sphingolipid long-chain base hydroxylation is important for growth and regulation of sphingolipid content and composition in Arabidopsis[END_REF]. Indeed, in a total extract of Arabidopsis seedlings, most of the GluCer containing d18 is associated with C16 FA to form d18/C16-GluCer. It is not true for GIPC which overall contains mostly t18 LCB even in C16-GIPC. The d18 LCB are thus channeled in C16 GluCer while t18 are mostly associated with VLCFA-GluCer and GIPC. The fact that in sbh mutants d18/C16 GluCer and GIPC are increased 10-fold, support the role of C4hydroxylation in channeling t18 species into VLCFA-complex sphingolipids. LCBs can also be unsaturated with 1 or 2 double bonds on sphinganine to produce d18:1, d18:2 or on phytosphingosine to produce t18:1, or t18:2. Desaturation can occur at C4 in trans configuration or on C8 in both cis and trans configurations. The enzymes responsible for these desaturations are the Δ4or Δ8-desaturases. Combination of these different LCB modifications (hydroxylation and desaturation) with their respective stereoisomers results in a subset of 9 different molecules (Figure 3). The Δ4-desaturase (DES4) was recently reported to be expressed in specific tissues, mainly pollen and at low level in flowers [START_REF] Michaelson | Functional characterization of a higher plant sphingolipid Delta4-desaturase: defining the role of sphingosine and sphingosine-1phosphate in Arabidopsis[END_REF]. The complete knockout of the DES4 does not lead to any developmental defect. However the des4 sphingolipidome of flower tissue revealed low levels of C16-GluCer, while VLCFA-GluCer and VLCFA-GIPC levels were unaffected, indicating that Δ4desaturated LCBs are preferentially combined with C16 rather than with longer fatty acyl chains. Interestingly, mutation of the Drosophila LCB desaturase homolog DES1 caused defective spermatogenesis in flies confirming the importance of unsaturated LCB in development [START_REF] Endo | Degenerative spermatocyte, a novel gene encoding a transmembrane protein required for the initiation of meiosis in Drosophila spermatogenesis[END_REF]. It has to be underlined that the C4-hydroxylation and Δ4-desaturation occur on the same site, suggesting that the respective enzymes may act in a competitive manner for their substrate. Interestingly, while Δ4-desaturation does not seem to be essential in Arabidopsis, it is prevalent in animal sphingolipids, leading to the formation of sphingosine (SPH), an important signal molecule (see below) (Figure 2). On the other hand, Δ8-desaturation, either in cis or trans configuration, is very common in plant sphingolipids [START_REF] Sperling | Are glucocerebrosides the predominant sphingolipids in plant plasma membranes?[END_REF]. The proportion of LCB and their cis/trans isomers is highly variable between plant species. For example, 20% of the total GluCer in Arabidopsis leaves is t18:1Δ8 E , while the t18:1Δ8 Z is only 3.4% of GluCer. In maize leaves, the levels of t:18:1 GluCer is low compared to Arabidopsis, furthermore the relative distribution of the E/cis and Z/trans are inverted with respectively 0.2% and 1% of total GluCer, reviewed by [START_REF] Pata | Plant sphingolipids: Decoding the enigma of the Sphinx[END_REF]. To date, the exact order in which C-4 hydroxylation, Δ4 and Δ8 desaturations occur is not yet fully defined, and the substrate specificities of each step remains poorly known. A possible sequence of the enzymatic activities along the pathway, as proposed by Pata et al., 2009 is shown in Figure 3.

Ceramide Synthesis and Inhibition, an Essential Component of Life Span

After LCB synthesis, the next step in sphingolipid biosynthetis involves the condensation of a LCB with a fatty acyl chain to form ceramide (Cer). The enzyme responsible for this reaction is the ceramide synthase (CerS). The CerS gene was originally isolated during a screen designed to identify genes that were differentially expressed cells that had undergone low or high number of divisions and that could thus be associated with longevity [START_REF] Egilmez | Specific alterations in transcript prevalence during the yeast life span[END_REF]. Like most cells, yeast cells are subjected to aging, which is defined as the total number of budding events (cell divisions). A gene, whose expression was high in early generations, decreased linearly during aging until being undetectable at final age (around 15 divisions), was characterized and named LAG1, for longevity assurance gene [START_REF] D'angelo | Vesicular and non-vesicular transport feed distinct glycosylation pathways in the Golgi[END_REF]. lag1 mutant displayed no defect in size, shape, budding pattern or rate of division; however, it exhibited increased life span corresponding to an average of 25 divisions [START_REF] D'angelo | Vesicular and non-vesicular transport feed distinct glycosylation pathways in the Golgi[END_REF]. Considering the negative correlation between LAG1 expression level and the increased life span, the author proposed that Lag1p could be directly or indirectly responsible for the average of 20-cell cycle limit in yeast life span. Another gene displaying a significant homology with LAG1 was found in yeast and named LAC1. The double knockout lag1/lac1 was either unviable or had severe growth defects depending on the strain background [START_REF] Barz | Two endoplasmic reticulum (ER) membrane proteins that facilitate ER-to-Golgi transport of glycosylphosphatidylinosi-tol-anchored proteins[END_REF]. Both proteins were localized in the ER and the viable double mutant combination displayed inability to transport GPIanchored proteins from the ER to the Golgi, a defect associated with sphingolipid levels [START_REF] Barz | Two endoplasmic reticulum (ER) membrane proteins that facilitate ER-to-Golgi transport of glycosylphosphatidylinosi-tol-anchored proteins[END_REF]. Indeed, LAG1 and LAC1 disruption resulted in reduced sphingolipid biosynthesis, because both genes were found to code for Acyl-CoA dependent ceramide synthases [START_REF] Guillas | C26-CoA-dependent ceramide synthesis of Saccharomyces cerevisiae is operated by Lag1p and Lac1p[END_REF][START_REF] Schorling | Lag1p and Lac1p are essential for the Acyl-CoAdependent ceramide synthase reaction in Saccharomyces cerevisae[END_REF].

Mammals, like humans or mice, have six different LAG1 homologs named LASS1-6 (for longevity assurance) that code for six different ceramide synthases (CerS1-6). The different enzymes display different substrate specificity, related in particular to the fatty acyl chain (Pewzner-Jung, [START_REF] Pewzner-Jung | When do Lasses (longevity assurance genes) become CerS (ceramide synthases)? Insights into the regulation of ceramide synthesis[END_REF][START_REF] Mizutani | Ceramide biosynthesis in keratinocyte and its role in skin function[END_REF] (Table 2). CerS3 is the only ceramide synthase utilizing a broad range of FA substrates, mainly C18 and C24, but also medium chains (C14, C16). Surprisingly, mammalian CerS, with the exception of CerS1, present an extra domain. This domain is a bona fide homeodomain except that it defines a very divergent subclass. [START_REF] Venkataraman | Do longevity assurance genes containing Hox domains regulate cell development via ceramide synthesis?[END_REF][START_REF] Riebeling | Two mammalian longevity assurance gene (LAG1) family members, trh1 and trh4, regulate dihydroceramide synthesis using different fatty acyl-CoA donors[END_REF][START_REF] Holland | Classification and nomenclature of all human homeobox genes[END_REF]. Although bona fide domain structure prediction studies suggest that they form three alpha helices in the correct conformation to bind DNA, no evidence for a DNA binding or transcription factor activity has been demonstrated yet [START_REF] Pewzner-Jung | When do Lasses (longevity assurance genes) become CerS (ceramide synthases)? Insights into the regulation of ceramide synthesis[END_REF]. However, ceramide synthases are ER transmembrane proteins and predicted to be inside the ER lumen. It is therefore unlikely that they could be in contact with DNA. Yeast and plants LAGs protein do lack this homeodomain.

The high number of CerSs in humans likely reflects tissue specific functions. CerS have been mainly characterized in cell cultures and a knockout model is only available for CerS2 (Table 2) [START_REF] Pewzner-Jung | A critical role for ceramide synthase 2 in liver homeostasis: I. alterations in lipid metabolic pathways[END_REF]. The cerS2 -/-mice could survive almost 2 years (like the wild type) but exhibited important liver dysfunction. Specifically, hepatocytes cerS2 - /-presented a shortened life span, with defective cellular regeneration that caused the development of liver carcinoma [START_REF] Pewzner-Jung | A critical role for ceramide synthase 2 in liver homeostasis: I. alterations in lipid metabolic pathways[END_REF]. CerS3 (Table 2) was found to be highly expressed in the skin [START_REF] Riebeling | Two mammalian longevity assurance gene (LAG1) family members, trh1 and trh4, regulate dihydroceramide synthesis using different fatty acyl-CoA donors[END_REF], which was correlated to the involvement of VLCFA-ceramides in the impermeable barrier function of the skin [START_REF] Coderch | Ceramides and skin function[END_REF]. LCFA-ceramide, namely C16-ceramide (Table 2), is the most abundant sphingolipid species in non-neuronal tissues, and its accumulation has been associated, in several cases, with the induction of apoptosis and cell stress [START_REF] Eto | C16 ceramide accumulates following androgen ablation in LNCaP prostate cancer cells[END_REF][START_REF] Osawa | Roles for C16-ceramide and sphingosine 1-phosphate in regulating hepatocyte apoptosis in response to tumor necrosis factor-alpha[END_REF][START_REF] Senkal | Alteration of ceramide synthase 6/C16-ceramide induces activating transcription factor 6-mediated endoplasmic reticulum (ER) stress and apoptosis via perturbation of cellular Ca 2+ and ER/Golgi membrane network[END_REF]. Tumor cells such as head and neck squamous carcinoma, melanoma and Lewis lung carcinoma, also contained high levels of C16-Cer [START_REF] Koybasi | Defects in cell growth regulation by C18:0-ceramide and longevity assurance gene 1 in human head and neck squamous cell carcinomas[END_REF] and the accumulation of such short species were associated with drug resistance and cell death [START_REF] Ponnusamy | Sphingolipids and cancer: ceramide and sphingosine-1-phosphate in the regulation of cell death and drug resistance[END_REF]. However, length of fatty acyl chain of sphingolipids could have opposite effects on carcinoma depending wether it occurred in the lung and colon carcinoma [START_REF] Hartmann | Long chain ceramides and very long chain ceramides have opposite effects on human breast and colon cancer cell growth[END_REF].

Our knowledge of the role of ceramide synthase activity was not only inferred from genetic studies, but also from the use of specific toxin-derived inhibitors. The fungus Fusarium moniliforme is a common contaminant of maize (Zea Mays) intended for human and animal consumption [START_REF] Marasas | Primary liver cancer and oesophageal basal cell hyperplasia in rats caused by Fusarium moniliforme[END_REF]. The presence of F. moniliforme in maize has been correlated with high incidence of human oesophageal cancer in southern Africa and outbreaks of leukoencephalomalacia in horses [START_REF] Pienaar | Field outbreaks of leukoencephalomalacia in horses consuming maize infected by Fusarium verticillioides (= F. moniliforme) in South Africa[END_REF][START_REF] Rabie | Moniliformin production and toxicity of different Fusarium species from Southern Africa[END_REF][START_REF] Gelderblom | Fumonisins: Novel mycotoxins with cancerpromoting activity produced by Fusarium moniliforme[END_REF]. The toxic and carcinogenic contaminants were isolated as Fumonisins (made by fusarium moniliforme) by [START_REF] Gelderblom | Fumonisins: Novel mycotoxins with cancerpromoting activity produced by Fusarium moniliforme[END_REF]. Fumonisin B1 (FB1) was found to be the most abundant and active fumonisin, inducing liver toxicity and cancer after 1 month of treatment in rats [START_REF] Gelderblom | Fumonisins: Novel mycotoxins with cancerpromoting activity produced by Fusarium moniliforme[END_REF]. The hepatotoxicity was correlated with both apoptosis and necrosis. Additionally, FB1 caused the appearance of foci of cell proliferation and carcinogenesis [START_REF] Gelderblom | Toxicity and carcinogenicity of the Fusarium moniliforme metabolite, fumonisin B1, in rats[END_REF][START_REF] Gelderblom | Toxicological effects in rats chronically fed low dietary levels of fumonisin B(1)[END_REF]. In vitro studies in rat hepatocytes revealed that FB1 targets sphingolipid biosynthesis by increasing LCB levels and reducing the ceramides and complex sphingolipids levels [START_REF] Yoo | Fumonisin inhibition of de novo sphingolipid biosynthesis and cytotoxicity are correlated in LLC-PK1 cells[END_REF][START_REF] Merrill | Fumonisin B1 inhibits sphingosine (sphinganine) N-acyltransferase and de novo sphingolipid biosynthesis in cultured neurons in situ[END_REF]. It was also shown that FB1 could directly inhibit the enzymatic activity of ceramide synthase in vitro, and the potency of the inhibition was correlated with the accumulation of free LCBs and acyl-chain substrates [START_REF] Merrill | Fumonisins and other inhibitors of de novo sphingolipid biosynthesis[END_REF]. Elevated level of sphingosine in urine was therefore used as a diagnostic indicator of FB1 consumption and the toxicity associated with altered sphingolipid metabolism.

In Arabidopsis, 3 LAG1 homologs are present in the genome and were named LOH1-3, for LAG One Homolog). Ceramides represent only 1-2% of total sphingolipids extract, as they are rapidly converted into more complex forms. Two major classes of ceramides exist in the Arabidopsis sphingolipidome: ceramide and hydroxyl-ceramides (hCer), the latter containing α-hydroxylated FAs [START_REF] Lynch | An introduction to plant sphingolipids and a review of recent advances in understanding their metabolism and function[END_REF][START_REF] Markham | Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry[END_REF]. αhydroxylation is however mostly found in complex sphingolipids downstream of ceramide (see below), suggesting that either only α-hydroxy-Cer pool is used for downstream biosynthesis of complex sphingolipids or that α-hydroxylation occurs on a fraction of ceramide pool and independently on the whole pool of complex sphingolipids. The biological role of α-hydroxylation is unknown, but it is tempting to speculate that it plays a structural role in mediating interactions between lipids or between lipids and proteins within the membrane layer. Arabidopsis mutants depleted in very long acyl chain sphingolipids were non-viable contrary mutants lacking long acyl chain sphingolipids demonstrating the importance of the length of acyl chain [START_REF] Markham | Sphingolipids containing very-long-chain fatty acids define a secretory pathway for specific polar plasma membrane protein targeting in Arabidopsis[END_REF]. VLCFA-sphingolipids were in particular involved in sorting specific material from the TGN towards the PM in vivo [START_REF] Markham | Sphingolipids containing very-long-chain fatty acids define a secretory pathway for specific polar plasma membrane protein targeting in Arabidopsis[END_REF]. VLCFA-sphingolipid depletion modified the dynamic of specific endomembrane compartments in particular for vesicle fusion during exocytosis and cytokinesis [START_REF] Molino | Inhibition of very long acyl chain sphingolipid synthesis modifies membrane dynamics during plant cytokinesis[END_REF].

Complex Sphingolipids in Plants

Downstream from ceramide synthase, several enzymes transfer a head group to the C1 of the ceramide backbone (Figure 1). Yeast, mammals and plants share a common head substitution but show also some specificity. In yeast, the IPC-synthase transfers a phosphorylinositol group from a phosphatidylinositol (PI) to ceramide to form Inositolphosphoryl-ceramide (IPC). In mammals, sphingomyelin synthases (SMS) transfer a phosphocholine from phosphatidylcholine to ceramide to produce sphingomuelin (SM), the most abundant mammalian sphingolipid. Apart from SM, mammals synthesize a neutral complex sphingolipid, glucosylceramide (GluCer) by transferring glucose to ceramide via glucosyceramide synthase (GCS). In plants, two major complex sphingolipids are found: a glucosylated ceramide (GluCer) and a glucosylated inositolphosphoryl-ceramide (GIPC). In Arabidopsis, GluCer-synthase (GCS) is encoded by a unique essential gene, and was recently shown to be important for Golgi morphology and protein secretion [START_REF] Melser | Glucosylceramide biosynthesis is involved in Golgi morphology and protein secretion in plant cells[END_REF].

Furthermore, there are three functional IPC-synthases [START_REF] Mina | Functional analyses of differentially expressed isoforms of the Arabidopsis inositol phosphorylceramide synthase[END_REF]. The first IPC-synthase identified was the gene product of ERH1 (enhancing RPW8-mediated HRlike cell death) [START_REF] Wang | An inositolphosphorylceramide synthase is involved in regulation of plant programmed cell death associated with defense in Arabidopsis[END_REF]. ERH1 was identified in a screening for mutations that enhanced hypersensitive response during pathogen infection (see below). The mutant erh1 did not display an obvious reduction in GIPC, probably because of redundancy with the two other IPC synthases, however it accumulated ceramides which are considered as the main cause of the increased cell death responses in the mutant.

GIPC and GluCer can be distinguished by their head groups as well as by their fatty acid and LCB contents. A complete sphingolipid profile of Arabidosis provided a detailed picture of the diversity of sphingolipids among the four classes of molecules; Cer, α-hydroxy-Cer (containing α-hydroxylated fatty acid), GluCer and GIPC [START_REF] Markham | Plant sphingolipids: Separation and identification of major sphingolipid classes from leaves[END_REF]. Four major observations can be made from these profiles. Firstly, t18 is the predominant form in both GluCer and GIPC, representing 90% of the total LCBs species. Secondly, t18 preferentially associates with VLCFA-Sphingolipids and d18 with LCFA-Sphingolipids. Thirdly, Cer can be α-hydroxylated or not (Cer versus hydroxy-Cer), while complex GluCer and GIPC only contain α-hydroxy FA. Finally, we have two major forms of GluCer: d18-LCFA(C16), t: 18-VLCFA(C24), and only one major form of GIPC: t:18 VLCFA(C24).

Sphingolipid distribution could also differ between different tissues suggesting specialized metabolic pathways. In plants, sphingolipids showed qualitative and quantitative variations according to the different tissues [START_REF] Islam | Sphingosine in plants: More riddles from the Sphinx?[END_REF][START_REF] Tellier | Comparative plant sphingolipidomic reveals specific lipids in seeds and oil[END_REF]. GIPCs and GluCer showed for instance quantitative variations between seedling and seed extracts however seeds but not oil showed specific accumulation of a new amine derived GIPC [START_REF] Tellier | Comparative plant sphingolipidomic reveals specific lipids in seeds and oil[END_REF]. The presence of specific sphingolipid species in specific tissues raises questions about their roles that need to be addressed.

LOCALISATION OF SPHINGOLIPID BIOSYNTHETIC ENZYMES AND SPHINGOLIPID TRANSPORT

Yeast IPCS, the human sphingomyelin synthase 1 (SMS1) and human GCS are all found in the Golgi [START_REF] Levine | Inositol phosphorylceramide synthase is located in the Golgi apparatus of Saccharomyces cerevisiae[END_REF][START_REF] Huitema | Identification of a family of animal sphingomyelin synthases[END_REF][START_REF] Tafesse | Both sphingomyelin synthases SMS1 and SMS2 are required for sphingomyelin homeostasis and growth in human HeLa cells[END_REF], while the human SMS2 is at the plasma membrane (PM) [START_REF] Yamaji | Sphingolipid metabolism and interorganellar transport: localization of sphingolipid enzymes and lipid transfer proteins[END_REF]. Interestingly in Arabidopsis, LOHs and GCS localize in the ER [START_REF] Marion | Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings[END_REF], IPCS-ERH1 has been instead localized at the trans Golgi network (TGN) [START_REF] Wang | An inositolphosphorylceramide synthase is involved in regulation of plant programmed cell death associated with defense in Arabidopsis[END_REF]. These different localizations indicate that the compartmentalization of the sphingolipid pathway and the channeling of ceramide into GluCer or IPC would depend on ceramide transport. Direct (non-vesicular) translocation of ceramide to the Golgi was proposed to occur thanks to ceramide transfer protein CERT (Cer transfer) [START_REF] Hanada | Molecular machinery for non-vesicular trafficking of ceramide[END_REF]. CERT contains a ceramide binding domain (START) and a plekstrin (PH) domain, needed for binding to PtdIP (phosphatidylinositolphospate) at the Golgi. It's integrity is essential for the synthesis of SM. Localization of a fluorescent Cer to the Golgi was also impaired in Hela cells expressing mutated CERT. CERT could also extract Cer from ER membranes in vitro demonstrating its role as ceramide carrier.

In Arabidopsis, a ceramide transfer protein has not been identified yet. However, a putative sphingosine-transfer protein has been isolated in a screening for mutants with accelerated cell death symptoms and named acd11 (for accelerated cell death) [START_REF] Brodersen | Knockout of Arabidopsis accelerated-cell-death11 encoding a sphingosine transfer protein causes activation of programmed cell death and defense[END_REF]. ACD11 is part of the GLTP protein family (glycolipid transfer protein) and at least two other homologs are present in the Arabidopsis genome, but the precise biochemical activity of these proteins awaits further work (Table 1).

SPHINGOLIPIDS ARE MAJOR MEMBRANE LIPIDS

Lipids are a vast class of molecules, which cover different cellular functions such as energy storage, protective barriers, membranes biogenesis and dynamics, and intracellular signaling.

The majority of lipids that build up a biological membrane are amphiphilic, with a hydrophilic head and a lipophilic tail. The polar head interacts with water while the hydrophobic tails tend to self-associate to avoid water, leading to the spontaneous formation of a bilayer which maximizes these hydrophilic/hydrophobic interactions. The propensities of lipids to self-assemble in simple or more complex structures could be one of the major mean of determining the different shapes found within the cells. Lipids can be organized as a flat surface for plasma membrane, spherical and the tubular structures in the vesicular system, and more intricate geometry in ER (endoplasmic reticulum) and Golgi [START_REF] Melser | Glucosylceramide biosynthesis is involved in Golgi morphology and protein secretion in plant cells[END_REF]. Sphingolipids play major roles in biological membrane and participate in membrane organization. Specific and diverse PM structures such as plasmodesmata of plant tissues or immunological synapsis of animal cells from the immunity system have been shown to need sphingolipids for both structural and functional meaning [START_REF] Gombos | Cholesterol and sphingolipids as lipid organizers of the immune cells' plasma membrane: their impact on the functions of MHC molecules, effector T-lymphocytes and T-cell death[END_REF][START_REF] Grison | Specific Membrane Lipid Composition Is Important for Plasmodesmata Function in Arabidopsis[END_REF]. Sphingolipids play essential roles in PM receptor clustering and signaling and malfunctioning of these processes have been associated with pathology. [START_REF] Lauwers | Association of yeast transporters with detergent-resistant membranes correlates with their cell-surface location[END_REF][START_REF] Lingwood | Lipid rafts as a membrane-organizing principle[END_REF][START_REF] Molino | Biogenesis and transport of membrane domains-potential implications in brain pathologies[END_REF].

The best way to look at the intracellular organization is to consider the possible routes followed by a plasma membrane (PM)-targeted protein after its synthesis in the ER. In a simplified view, after synthesis in the ER membranes a PM-protein usually reaches the Golgi apparatus. The Golgi is composed of a cis part which is in continuity with ER membranes, a medial, and a trans part which constitutively delivers proteins to all post-Golgi compartments, including the plasma membrane, the different endosome populations, and the prevacuolar and vacuolar compartments. Such a step by step transport defines the eukaryotic secretory pathway (Figure 4).

Table 1. Enzymes and genes from Arabidopsis thaliana involved in sphingolipid biosynthesis Table 1. (Continued)

Subcellular purification of different intracellular membranes has revealed different lipid composition [START_REF] Moreau | Lipid trafficking in plant cells[END_REF][START_REF] Warnecke | Recently discovered functions of glucosylceramides in plants and fungi[END_REF]. In particular, phospholipids are the most abundant species in membranes, whereas sphingolipids and sterols can be very low or absent (such as in the ER membranes), or representing up to 25% each of total lipid levels (as in the plasma membrane). Apart from the plasma membrane, sphingolipids are enriched also in the late compartments and in the plant tonoplast, where they represent 10-20% of the total membrane lipids (Figure 4) [START_REF] Moreau | Lipid trafficking in plant cells[END_REF][START_REF] Warnecke | Recently discovered functions of glucosylceramides in plants and fungi[END_REF]. Golgi and late secretory compartments contain discrete amounts of both sterols and sphingolipids, with a composition showing more similarity to the PM than to the ER membranes. Membrane lipids appear thus compartmentalized in the cell with a specific gradient of sphingolipid and sterol along the secretory pathway with the highest accumulation at the PM. Interestingly, while sphingolipids are enriched in the late compartments of the secretory pathway, most of the biosynthesis is localized to the ER (Table 1). There are however few exceptions with the glycosylation of Ceramide to form GlcCer that occurs in the cytosolic leaflet of early Golgi cisternae. Once formed, GluCer is the transported across the Golgi either via a vesicular pathway or with the lipid transfer protein known as FAPP2 (D'Angelo, [START_REF] D'angelo | Vesicular and non-vesicular transport feed distinct glycosylation pathways in the Golgi[END_REF]. Interestingly, these two differently transported GlcCer pools follow two different glycosylation mechanisms, and in different Golgi subdomains. In particular, GlcCer transported by direct protein transfer is used to synthesize glycosphingolipids known as globosides in the TGN while vesicular transported GlcCer is used in the Golgi cisternae for ganglioside production, such as that of GM3 (D'Angelo, [START_REF] D'angelo | Vesicular and non-vesicular transport feed distinct glycosylation pathways in the Golgi[END_REF].

Table 2. Ceramide synthases

SPHINGOLIPID HOMEOSTASIS IN CELL GROWTH

Pools of different LCBs, ceramide and complex sphingolipids are highly dynamic. In mammals, activation of sphingomyelinase and ceramidase resulted in the accumulation of ceramide and LCBs respectively, both involved in growth arrest and cell death [START_REF] Mao | Ceramidases: regulators of cellular responses mediated by ceramide, sphingosine, and sphingosine-1-phosphate[END_REF]. Several studies indicateed that high levels of LCBs (especially sphingosine, SPH) and ceramides were associated with growth arrest, while phosphorylated sphingosine (S1P) induced cell proliferation [START_REF] Ping | Ceramide can induce cell death in sensory neurons, whereas ceramide analogues and sphingosine promote survival[END_REF]. Thus, the relative level of ceramide and SPH versus S1P seems to associate with opposite cellular effects leading to the idea that sphingolipids may act as a rheostat system regulating the cellular switch between survival or death, reviewed by [START_REF] Hannun | Principles of bioactive lipid signalling: Lessons from sphingolipids[END_REF].

Catabolism of complex sphingolipids is better understood in humans, and several autosomal recessive mutations have been reported and classified as sphingolipidosesbecause they lead to sphingolipid accumulation. Degradation of ceramide is achieved by ceramidase, sphingomylein by sphingomyelinase, glucosylceramide by glucocerebrosidase, and galactosylceramide by galactosylceramidases. Mutations of glucosylceramidase induce the Gaucher disease, which is the most common lysosomal storage disease. In patients with Gaucher disease, GluCer accumulates in spleen, liver, kidneys, lungs and brain, eventually causing organ degeneration [START_REF] Grabowski | Acid beta-glucosidase: enzymology and molecular biology of Gaucher disease[END_REF]. Genetic defects of sphingomyelinase causes Niemann-Pick type A and B diseases, characterized by the accumulation of sphingomyelin and hepatic and neuronal degeneration [START_REF] Schuchman | Acid sphingomyelinase, cell membranes and human disease: lessons from Niemann-Pick disease[END_REF]. Ceramidase mutations cause the Faber disease, characterized by progressive body deformation and, 20 pain because of ceramide accumulation in joints and the development of granulomas [START_REF] Ozkara | Recent advances in the biochemistry and genetics of sphingolipidoses[END_REF][START_REF] Molino | Biogenesis and transport of membrane domains-potential implications in brain pathologies[END_REF].

In plants the degradative enzymes for GluCer and GIPC (analog of sphingomyelinase) have not yet been found. Three putative ceramidases (CDase) are present in the Arabidopsis genome (Table 1), but they have not been characterized yet. The only plant ceramidase so far characterized is the rice OsCDase1. It localizes to the ER and Golgi and can hydrolyze a fluorescent ceramide analog in vitro [START_REF] Pata | Molecular Cloning and Characterization of OsCDase, a Ceramidase Enzyme from Rice[END_REF]. However, no ceramidase activity against endogenous Cer was detected. On the contrary, OsCDase1 expression in yeast CDases knockout induced an increase of C-26-ceramide. This observation suggested that OsCDase1, displays a reverse ceramidase activity, resulting in an Acyl-CoA independent ceramide synthesis, similar observation where made for yeast YPC1p and YDC1p [START_REF] Jiang | Suppressor analysis points to the subtle role of the LAG1 ceramide synthase gene in determining yeast longevity[END_REF].

The balance between ceramide synthesis and degradation appears to be essential for the homeostasis of the three signaling molecules Cer, SPH and S1P. Several stresses induce degradation of sphingomyelin and accumulation of ceramide. Known factor that have been associated with ceramede accumumation are: proapoptotic cytokine (TNF-α, IL-1β, FAS ligand, interferon γ), chemotherapeutic agents that induces DNA damage-mediated apoptosis, UV, ionizing irradiation and serum deprivation [START_REF] Mao | Ceramidases: regulators of cellular responses mediated by ceramide, sphingosine, and sphingosine-1-phosphate[END_REF]. Sphingosine and ceramide have been reported to induce both cell growth arrest and apoptosis [START_REF] Pettus | The sphingosine kinase 1/sphingosine-1-phosphate pathway mediates COX-2 induction and PGE2 production in response to TNF-alpha[END_REF], [START_REF] Futerman | The complex life of simple sphingolipids[END_REF]. In particular, in several carcinogenic cell lines, SPH was found to be an efficient inducer of cell death. SPH inhibits protein kinase C (PKC), the tyrosine kinase SRC (sarcoma), several Ca ++ -Calmodulin dependent kinases [START_REF] Jefferson | Sphingosine inhibits calmodulin-dependent enzymes[END_REF][START_REF] Igarashi | Effect of chemically well-defined sphingosine and its N-methyl derivatives on protein kinase C and src kinase activities[END_REF] and mitogen activated kinases ERK1/2 [START_REF] Jarvis | Coordinate regulation of stress-and mitogenactivated protein kinases in the apoptotic actions of ceramide and sphingosine[END_REF]. Apart from targeting cytosolic proteins, SPH also alters membrane systems. High levels of SPH increase permeability of lysosomes [START_REF] Kagedal | Sphingosine-induced apoptosis is dependent on lysosomal proteases[END_REF] and mitochondria [START_REF] Cuvillier | Sphingosine 1-phosphate antagonizes apoptosis of human leukemia cells by inhibiting release of cytochrome c and Smac/DIABLO from mitochondria[END_REF] which respectively release cathepsins and Cytochrome C to the cytosol that in turn induces apoptosis. Treatment of Hela cells with C-6 ceramide induces endogenous increase of SPH followed by Golgi fragmentation and apoptosis [START_REF] Hu | Golgi fragmentation is associated with ceramide-induced cellular effects[END_REF]. Overexpression of ceramidases also caused SPH increases and a similar effect on Golgi fragmentation leading to apoptosis [START_REF] Markham | Plant sphingolipids: Separation and identification of major sphingolipid classes from leaves[END_REF].

SPHINGOLIPIDS ARE ASSOCIATED WITH PATHOGEN INFECTION IN PLANTS AND ANIMALS

Sphingolipids play roles in signal transduction pathways regulating stress responses in eukaryotes. In plants LCB modifications including phosphorylation as well as unsaturation were shown to take part in the tolerance to environmental extreme and responses to chilling [START_REF] Chen | Sphingolipid Delta8 unsaturation is important for glucosylceramide biosynthesis and low-temperature performance in Arabidopsis[END_REF][START_REF] Dutilleul | Phytosphingosine-phosphate is a signal for AtMPK6 activation and Arabidopsis response to chilling[END_REF]. In the other side, modulation of the sphingolipid metabolism was associated with responses to heat stress in yeast [START_REF] Sun | Orm protein phosphoregulation mediates transient sphingolipid biosynthesis response to heat stress via the Pkh-Ypk and Cdc55-PP2A pathways[END_REF].

Several Arabidopsis mutations modifying sphingolipid homeostasis enhanced sensitivity to pathogen infections. Upon infection, plant responses include generation of H 2 O 2 at fungal penetration sites and triggering of the hypersensitive response (HR). HR is a form of programmed cell death at the infection site [START_REF] Levine | H 2 O 2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response[END_REF]. The gene RPW8 confers resistance to powdery mildew, a disease caused by several fungi targeting hundreds of dicotyledonous plants. In Arabidopsis, the Columbia (Col 0) accession does not have a RPW8 gene while WS accession contains two RPW8 copies. EMS mutagenesis on a Col 0 background overexpressing RPW8 enabled to find mutations that suppress the resistance conferred by RPW8 [START_REF] Wang | An inositolphosphorylceramide synthase is involved in regulation of plant programmed cell death associated with defense in Arabidopsis[END_REF]. Such a screen identified the plant IPCsynthase ERH1, as already mentioned, erh1 mutation prompted the accumulation of high level of ceramides explaining the spontaneous induction of cell death. Similarly, the ceramide kinase ACD5 (for accelerated cell death) [START_REF] Brodersen | Knockout of Arabidopsis accelerated-cell-death11 encoding a sphingosine transfer protein causes activation of programmed cell death and defense[END_REF], as well as a sphingosine transfer protein ACD11 [START_REF] Liang | Ceramides modulate programmed cell death in plants[END_REF], were identified in a screen for mutations that cause HR responses in the absence of pathogens. Altogether these observations indicated that any mutation resulting in increased levels of ceramide, possibly at the ER, induces cell growth arrest and PCD in plant as in mammals. Ceramide phosphorylation, or its transport out of the ER, seems to be protective factors against HR, since they would prevent Cer accumulation [START_REF] Liang | Ceramides modulate programmed cell death in plants[END_REF].

As already mentioned, fungal infection was shown to directly modify Cer levels by inhibition of ceramide synthase activity. Mycotoxins such as AAL and FB1 inhibit CerS and induce cell death. Several pieces of evidence suggested that in plants, the bioactive molecules leading to cell death are accumulated free LCBs. AAL induced death symptoms can be suppressed by concomitant pharmacological treatment with Myriocin, an inhibitor of the SPT [START_REF] Spassieva | The plant disease resistance gene Asc-1 prevents disruption of sphingolipid metabolism during AAL-toxin-induced programmed cell death[END_REF], which abolished free LCB accumulation. Furthermore an lcb1 mutant, impaired in SPT activity, was not able to accumulate free LCBs and was resistant to FB1 treatment. Application of exogenous LCBs (d18:0, t:18:0, t:18:1) induced ROS (reactive oxygen species) production and pro-death responses [START_REF] Shi | Rho kinase in the regulation of cell death and survival[END_REF]. The LCB-dependent induction of cell death was found to be dependent upon nuclear Ca ++ release [START_REF] Lachaud | Nuclear calcium controls the apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells[END_REF]. Studies in tobacco on the role of sphingolipids in plant immunity against mycotoxins suggest that LCBs modulate the salicylic acid-dependent responses (Rivas-San Vicente, [START_REF] Rivas-San | Disruption of sphingolipid biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f. sp. lycopersici[END_REF]. Finally, a MYB transcription factor (AtMYB30), a positive regulator of cell death in plants during HR responses, has been shown to directly increase the transcription of the genes encoding for the 4 core enzyme of the elongase complex. The increase of VLCFA elongation would directly impact on VLCFAbearing lipids among them the sphingolipids, as demonstrated by [START_REF] Raffaele | A MYB Transcription Factor Regulates Very-Long-Chain Fatty Acid Biosynthesis for Activation of the Hypersensitive Cell Death Response in Arabidopsis[END_REF]. The authors suggested that acyl chain length would be a major determinant in modulating cell death and defense responses. Alternatively VLCFA modifications could alter pathogen recognition at the membrane by modifying structural properties of plasma membrane lipids. It can also not be excluded that the increase of VLCFA simply enhanced accumulation of epicuticular waxes and increase the epidermal physical barriers preventing pathogen entry.

Interestingly enough, human viruses are known to use membranes and their components (including sphingolipids) in different steps of their life cycle including attachment, intracellular transport, and budding, reviewed in(Schneider-Schaulies and Schneider-Schaulies 2015). Examples for sphingolipid-dependent virus entry are found for human immunodeficiency virus (HIV), which besides its protein receptors also interacts with glycosphingolipids (GSLs) [START_REF] Sorice | Evidence for cell surface association between CXCR4 and ganglioside GM3 after gp120 binding in SupT1 lymphoblastoid cells[END_REF] as well as the rhinovirus, which promotes the formation of ceramide-and glycosphingolipids-enriched platforms [START_REF] Dreschers | Infections with human rhinovirus induce the formation of distinct functional membrane domains[END_REF]. Furthermore viruses can also manipulate sphingolipid metabolism as for example influenza A virus, which activates sphingosine kinase 1 and the transcription factor NF-κB [START_REF] Seo | Sphingosine kinase 1 serves as a pro-viral factor by regulating viral RNA synthesis and nuclear export of viral ribonucleoprotein complex upon influenza virus infection[END_REF].

SPHINGOLIPID PHOSPHORYLATION: THE SPHINGOSINE-P PARADIGM IN ANIMALS AND PLANTS

Phosphorylated LCBs (LCB-P) and corresponding kinases have been so far identified in all organisms. In yeast reversible phosphorylation is carried out by two LCB-kinases, LCB4p and LCB5p [START_REF] Nagiec | The LCB4 (YOR171c) and LCB5 (YLR260w) genes of Saccharomyces encode sphingoid long chain base kinases[END_REF] while dephosphorylation is carried out by the LCB phosphatases LCB3p and YSR3p (Figure 5). LCB-P can also be degraded by the LCB phosphate-lyase DPL11p, which irreversibly breaks down the LCBs-P in ethanolaminephosphate and a hexadecanal aldhyde (Figure 5). While LCB3p, YSR3p and DPL1p are localized to perinuclear and cortical ER, LCB4p and LCB5p are found only at the cortical ER and Golgi [START_REF] Hait | Lcb4p sphingoid base kinase localizes to the Golgi and late endosomes[END_REF][START_REF] Iwaki | Intracellular trafficking pathway of yeast long-chain base kinase Lcb4, from its synthesis to its degradation[END_REF]. Single mutant strains for any of these LCB-P related genes resulted in wild type phenotype. On the contrary double mutant dpl1/lcb3 was not viable but could be rescued by lcb4 mutation in the triple dpl1/lcb3/lcb4 mutant [START_REF] Nagiec | The LCB4 (YOR171c) and LCB5 (YLR260w) genes of Saccharomyces encode sphingoid long chain base kinases[END_REF]. LCB-P accumulation was thus correlated with growth inhibition (Figure 5), which was confirmed by LCB4 or LCB5 overexpression in dpl1lcb3/lcb4 mutant that increased LCB-P levels and arrested growth [START_REF] Nagiec | The LCB4 (YOR171c) and LCB5 (YLR260w) genes of Saccharomyces encode sphingoid long chain base kinases[END_REF]. A number of studies suggested that LCB-P could also have a role in several stress responses in yeast. For example heat stress induced LCB-P increase and lcb3 mutants, who accumulate LCB-P, survived better upon heat shock, while lcb3 overexpression resulted in lower LCB-P and higher sensitivity to temperature [START_REF] Mandala | Sphingoid base 1-phosphate phosphatase: A key regulator of sphingolipid metabolism and stress response[END_REF]. Altogether these observations suggested that LCB-P plays a dual role according to its concentration in the cell. A moderated increase of LCB-P would correlate with a protective role during stress responses, while greater accumulation of LCB-P would correlate with growth arrest.

It has been reported that G1 cell cycle arrest induced by rapamycin involved lcb4p degradation [START_REF] Iwaki | Intracellular trafficking pathway of yeast long-chain base kinase Lcb4, from its synthesis to its degradation[END_REF]. Thus, it is possible that LCB-Ps are also involved in the maintenance of G1 arrest. A similar function was proposed for the mammalian S1P. In mammals, overexpression of SPHK1, a homologue of Lcb4p, results in a decreased number of cells in G0/G1 and an increased number of cells in S phase, suggesting that LCB-P could be involved in G1-to-S transition [START_REF] Jenkins | Role for de novo sphingoid base biosynthesis in the heat-induced transient cell cycle arrest of Saccharomyces cerevisiae[END_REF].

In Arabidopsis, four LCBs kinases have been identified: Sphk1 and sphk2 (for sphingosine kinase 1 and 2), and LCBK1, LCBK2 (for LCB kinase 1 and 2) (Table 1). They have different subcellular localizations involving tonoplast, mitochondria and PM (Table 1). LCBs-P can be dephosphorylated by the phosphatases SPPase (sphingosine phosphatase) and hydrolyzed by the unique lyase atDPL1 in the ER [START_REF] Tsegaye | Arabidopsis mutants lacking long chain base phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-18:1 long chain base phosphate[END_REF]. Arabidopsis dpl1 mutant does not accumulate much LCB-P or LCB. This observation suggests that LCB-P degradation via AtDPL1 does not represent a major pathway to control LCB/LCB-P levels in normal growth conditions. However, LCB and LCB-P accumulation are associated with stress conditions and cell death. The Arabidopsis dpl1 mutant showed a hypersensitive response to FB1, which correlates with an accumulation of both LCB and LCB-P. Thus, AtDPL1 activity seems to become important in situations of stress leading to high LCB accumulation. It has to be noted that phosphorylated LCBs cannot be directly incorporated into ceramide, but can be reversibly dephosphorylated into LCB and then incorporated into sphingolipids, providing a different pathway to control LCB-P accumulation. Sphingosine phosphate (S1P) has been associated with cell proliferation in animals. Originally, S1P was shown to induce mitosis in fibroblasts [START_REF] Zhang | Sphingosine-1-phosphate, a novel lipid, involved in cellular proliferation[END_REF]. Later, the pro-mitotic activity was associated with increases in Ca ++ release via a non-identified mechanism which is phosphatidyl-inostol-1,4,5-P-independent [START_REF] Mattie | Sphingosine-1-phosphate, a putative second messenger, mobilizes calcium from internal stores via an inositol trisphosphate-independent pathway[END_REF]. Numerous studies then revealed that S1P could directly interact at the plasma membrane with several G protein coupled receptors (GPCR), leading to their activations. At least five different S1P-GPCR mechanism mediating proliferative responses have been described in animals [START_REF] Mao | Ceramidases: regulators of cellular responses mediated by ceramide, sphingosine, and sphingosine-1-phosphate[END_REF][START_REF] Rosen | Sphingosine 1-phosphate receptor signaling[END_REF] that involve angiogenesis, vascular tissue development, immune responses and neuronal development. In human cell lines, S1P was also found to bind to and modulate the activity of a histone deacetylase in the nucleus [START_REF] Hait | Regulation of histone acetylation in the nucleus by sphingosine-1-phosphate[END_REF]) and a E3 Ubiquitin ligase complex of tumor necrosis factor receptor associated factor 2 (TRAF2) in the cytosol [START_REF] Alvarez | Sphingosine-1-phosphate is a missing cofactor for the E3 ubiquitin ligase TRAF2[END_REF]). S1P signaling system was also associated with several physiological process as well as human pathology especially inflammation process and inflammatory disease, immunity cells physiology [START_REF] Garris | Sphingosine-1-phosphate receptor 1 signalling in T cells: trafficking and beyond[END_REF][START_REF] Gonzalez-Cabrera | S1P signaling: new therapies and opportunities[END_REF] as well as cancer [START_REF] Zhang | Sphingosine kinase 1 and cancer: a systematic review and meta-analysis[END_REF].

In plants, the pharmacological treatment with S1P induces stomata closure [START_REF] Ng | Drought-induced guard cell signal transduction involves sphingosine-1-phosphate[END_REF]. The S1P effect was abolished in G protein mutant, indicating that S1P activity is dependent on G protein-coupled receptor activity in plants [START_REF] Coursol | Sphingolipid signalling in Arabidopsis guard cells involves heterotrimeric G proteins[END_REF]. The role of S1P was confirmed by the fact that the sphk1 mutant or SPHK1 overexpression showed respectively lower and higher sensitivity to stomatal opening after abscissic acid stimulation [START_REF] Worrall | Involvement of sphingosine kinase in plant cell signalling[END_REF]. The activity of the SPHK1 is correlated with stomatal sensitivity to ABA. ABA inhibition of seed germination was also much less pronounced in sphk1 mutants. However the analysis of Arabidopsis Δ4-desaturase mutant lacking both sphingosine and S1P revealed no developmental defect (including stomata closure) [START_REF] Michaelson | Functional characterization of a higher plant sphingolipid Delta4-desaturase: defining the role of sphingosine and sphingosine-1phosphate in Arabidopsis[END_REF]. The latest observation strongly suggests that another mechanism may be responsible for stomata closure [START_REF] Lynch | Lipid signaling in Arabidopsis: no sphingosine? No problem![END_REF]. Since SPHK1 seems to use a variety of LCBs as substrate, different free LCBs than S1P could be responsible for ABA-related responses.

REGULATION OF SPHINGOLIPID METABOLISM

The homeostasis of the different sphingolipids needs to be tightly controlled to ensure cell viability. While in plants the regulation of key enzymes of the pathway remains largely unknown, in animals and yeast several important regulatory mechanisms have been uncovered.

An important breakthrough in the understanding of homeostasis and regulation of sphingolipid metabolism was recently achieved thanks to a global yeast genetic interaction study aiming to understand the role of ORM proteins, which are involved in several human inflammatory diseases. There are 2 ORM genes (ORM1 and 2) in Arabidopsis, yeast and three in humans [START_REF] Han | Orm1 and Orm2 are conserved endoplasmic reticulum membrane proteins regulating lipid homeostasis and protein quality control[END_REF][START_REF] Breslow | Sphingolipid homeostasis in the endoplasmic reticulum and beyond[END_REF][START_REF] Kimberlin | Arabidopsis 56-amino acid serine palmitoyltransferase-interacting proteins stimulate sphingolipid synthesis, are essential, and affect mycotoxin sensitivity[END_REF]. They are ER-localized proteins involved in protein folding and unfolding responses [START_REF] Han | Orm1 and Orm2 are conserved endoplasmic reticulum membrane proteins regulating lipid homeostasis and protein quality control[END_REF]. ORM proteins were found to be negative regulators of the serine palmitoyl transferase (SPT), the first step of sphingolipid biosynthesis [START_REF] Breslow | Orm family proteins mediate sphingolipid homeostasis[END_REF]. Characterization of ORM-SPT complex allowed the isolation of SAC1, a phosphoinositide phosphatase involved in protein secretion in response to nutrients in both animals and yeast [START_REF] Rohde | The tor pathway regulates gene expression by linking nutrient sensing to histone acetylation[END_REF][START_REF] Blagoveshchenskaya | SAC1 lipid phosphatase and growth control of the secretory pathway[END_REF]. Interestingly, while both SAC1 and ORMs have a negative effect on SPT, they bind SPT independently, suggesting that they probably act as two independent regulatory mechanisms. Conversely, yeast orm1/2 presented an increased SPT activity and an accumulation of LCB but not ceramides. This observation suggests that LCB and ceramide pools are not simply interconnected and in balance, but that CerS activity is also tightly regulated [START_REF] Breslow | Orm family proteins mediate sphingolipid homeostasis[END_REF]. In Arabidopsis, small subunits of SPT (ssSPT) also limit sphingolipid biosynthesis and therefore modulate FB1 sensitivity and are essential for male gametophyte viability [START_REF] Kimberlin | Arabidopsis 56-amino acid serine palmitoyltransferase-interacting proteins stimulate sphingolipid synthesis, are essential, and affect mycotoxin sensitivity[END_REF].

The activity of ORM and its inhibitory effect on SPT depends upon ORM hyperphosphorylation. Interestingly, ORM hyperphosphorylation is also sensitive to ceramide synthesis. In yeast ceramide synthase mutant lac1, ORMs phosphorylation was increased and SPT activity decreased [START_REF] Breslow | Orm family proteins mediate sphingolipid homeostasis[END_REF]. This important observation suggests that ceramide levels could regulate ORM activity, defining a negative feedback loop that prevents ceramide accumulation.

In yeast, ceramide synthase activity requires another cofactor, LIP1 (for lag1/lac1 interacting protein), essential for CerS activity. LIP1 copurified with Lag1p and Lac1p [START_REF] Vallee | Lip1p: A novel subunit of acyl-CoA ceramide synthase[END_REF]. In vivo labeling experiments showed that lip1 was impaired in ceramide synthesis, also with a sphingolipid profile similar to lac1/lag1. The exact mechanism by which LIP1 works is not clear but it has been suggested that LIP1 could be important for specifying ceramide synthase substrate specificities. In fact, the purification of ceramide synthase complex revealed several protein associations: two Lip1p proteins could bind Lag1p-Lac1p heterodimers or Lag1p/Lac1p homodimers [START_REF] Vallee | Lip1p: A novel subunit of acyl-CoA ceramide synthase[END_REF]. Thus, the different complexes could have different enzymatic activities, i.e., different specificities for acyl chain length or LCB.

Plant and animal genomes do not seem to encode any LIP1p related genes (data not shown). However, a different mechanism involving enzymatic channeling could regulate sphingolipid biosynthesis. It was recently demonstrated that the VLCFA-ceramide synthase CerS2, but not the LCFA-CerS4, interacted specifically with elongase enzymes allowing efficient incorporation of very long acyl chain-CoA into sphingolipids [START_REF] Ohno | ELOVL1 production of C24 acyl-CoAs is linked to C24 sphingolipid synthesis[END_REF]. In yeast, the target of rapamycin complex 2 (TORC2) was recently found to control de novo ceramide synthesis [START_REF] Aronova | Regulation of ceramide biosynthesis by TOR complex 2[END_REF]. Arnova et al. isolated a conditional allele with a mutation in AVO3 (avo3), a subunit of the TORC2 complex that grows normally at 25°C, but slows down at 30°C. The authors found that a few hours after transfer to 30° the synthesis of ceramides was strongly reduced. Furthermore the overexpression of a target of TOR, the kinase YPK2, reverted avo3 phenotypes and ceramide depletion, suggesting that YPK2 kinase activity is acting in the pathway downstream of TOR and upstream of ceramide synthase. Another interesting result regarded calcineurin, a conserved Ca++/Calmodulin-regulated phosphatase that inhibits several targets of TOR. Deletion of regulatory subunit of the calcineurin could also restore proper ceramide synthesis in avo3 [START_REF] Aronova | Regulation of ceramide biosynthesis by TOR complex 2[END_REF].

In conclusion, TOR is a kinase involved in stress sensing in all eukaryotes and a master regulator acting upstream Orms proteins in the signaling cascade regulating sphingolipids metabolism [START_REF] Liu | Regulation of sphingolipid synthesis through Orm1 and Orm2 in yeast[END_REF]. Such a master regulator also controls actin polarization and endocytosis via unclarified mechanisms [START_REF] Rispal | Target of rapamycin complex 2 regulates actin polarization and endocytosis via multiple pathways[END_REF]. Sphingolipids were also shown to be required for endocytosis and actin organization [START_REF] Zanolari | Sphingoid base synthesis requirement for endocytosis in Saccharomyces cerevisiae[END_REF].

Interestingly enough, processes that maintain lipid and protein clustering and thus the formation of membrane domains have been proposed to be involved in cortical actin [START_REF] Goswami | Nanoclusters of GPI-anchored proteins are formed by cortical actin-driven activity[END_REF]. Thus, it is plausible that the control of membrane lipid biosynthesis is a basic process regulating membrane composition, tension and organization necessary for different plasma membrane activities, as well as intracellular trafficking pathways.

Figure 1 .

 1 Figure 1. Sphingolipid structure and diversity. A common ceramide backbone is shared among the different organisms. Ceramide has two moieties: a Long Chain Base (LCB) and an N-linked fatty acid. Polar head groups (left) and fatty acyl chain length (right) are indicated for Arabidopsis, Saccharomyces cerevisiae and humans. Ceramide structure is from Pata et al., 2010.

Figure 2 .

 2 Figure 2. Sphingoid base biosynthesis. SPT for serine palmitoyl transferase. KCR for keto sphinganine reductase. d18:0, t18:0, d18:1 nomenclature indicates the number of -OH (di or tri) and the number of double bound. Predominant LCBs species in animals and plants are indicated.

Figure 3 .

 3 Figure 3. LCBs modifications. Possible sequential order of LCB modifications. The pathway does not take into account the different possible isomers.

Figure 4 .

 4 Figure 4. Sphingolipids distribution in plant secretory pathway. Model underlining the sphingolipid distribution along the plant secretory pathway (in gray). Plant Cer, GluCer, and GIPC localization is shown, according to the localization of biosynthetic enzymes. Arrows consider the possible vesicular transport of sphingolipid (grey arrows and question marks).

Figure 5 .

 5 Figure 5. LCB phosphorylation pathway. LCBs can be phosphorylated and dephosphorylated by sphingosine-or LCB-kinases and sphingosine-phosphatase (SPPase) respectively. Only phosphorylated LCBs are substrate of degradation by the lyase DPL1. The interconnection of the phosphorylation pathway with the ceramide synthase is shown. In boxes, molecule functions in different organisms have been indicated. Interrogation points represent speculations.