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The lipid composition of organelles acts as a landmark to define membrane identity and specify subcellular function. Phosphoinositides are anionic lipids acting in protein sorting and trafficking at the trans-Golgi network (TGN). In animal cells, sphingolipids control the turnover of phosphoinositides through lipid exchange mechanisms at endoplasmic reticulum/TGN contact sites. In this study, we discover a mechanism for how sphingolipids mediate phosphoinositide homeostasis at the TGN in plant cells. Using multiple approaches, we show that a reduction of the acyl-chain length of sphingolipids results in an increased level of phosphatidylinositol-4-phosphate (PtdIns(4)P or PI4P) at the TGN but not of other lipids usually coupled to PI4P during exchange mechanisms. We show that sphingolipids mediate Phospholipase C (PLC)-driven consumption of PI4P at the TGN rather than local PI4P synthesis and that this mechanism is involved in the polar sorting of the auxin efflux carrier PIN2 at the TGN. Together, our data identify a mode of action of sphingolipids in lipid interplay at the TGN during protein sorting., Lipid composition impacts the function of cellular membranes. Here the authors show that a reduction in sphingolipid acyl-chain length promotes phosphoinositide consumption by phospholipase C at the Arabidopsis trans-Golgi network which in turn regulates sorting of the auxin efflux carrier PIN2.

International audience; The Plasma Membrane (PM) is a key structure protecting the cell, regulating nutrient exchanges and acting as a control tower allowing the cell to perceive signals. Plasma comes from the greek πλάσμα meaning "which molds", meaning that the PM takes the shape of the cell by delimitating it. The PM harbors the appropriate signaling cascades allowing adaptive responses ensuring proper cell functions in a continuously fluctuating environment, crucial for cell survival. To address this challenge, the PM needs to be both stable and robust yet incredibly fluid and adaptable. This amazing combination of long-term stability and short-term dynamics in order to adapt to signals relies on its fascinating molecular organization. PMs are extremely complex systems, harboring many different molecular species of lipids in which heterogeneity is more likely to occur than homogeneity. In plants as in animals, the recent development of proteomics, lipidomics and methods to visualize lipids and proteins in vivo has greatly increased our knowledge of the PM. Corresponding Author Sébastien Mongrand

International audience; During early seedling development, the shoot apical meristem is protected from damage as the seedling emerges from soil by the formation of apical hook. Hook formation requires differential growth across the epidermis below the meristem in the hypocotyl. The plant hormones ethylene and auxin play key roles during apical hook development by controlling differential growth. We provide genetic and cell biological evidence for the role of ADP-ribosylation factor 1 (ARF1)-GTPase and its effector ARF-guanine-exchange factors (GEFs) of the Brefeldin A-inhibited GEF (BIG) family and GNOM in ethylene-and auxin-mediated control of hook development. We show that ARF-GEF GNOM acts early, whereas BIG ARF-GEFs act at a later stage of apical hook development. We show that the localization of ARF1 and BIG4 at the trans-Golgi network (TGN) depends on ECHIDNA (ECH), a plant homolog of yeast Triacylglycerol lipase (TLG2/SYP4) interacting protein Tgl2-Vesicle Protein 23 (TVP23). BIGs together with ECH and ARF1 mediate the secretion of AUX1 influx carrier to the plasma membrane from the TGN during hook development and defects in BIG or ARF1 result in insensitivity to ethylene. Thus, our data indicate a division of labor within the ARF-GEF family in mediating differential growth with GNOM acting during the formation phase whereas BIGs act during the hook maintenance phase downstream of plant hormone ethylene.

Cell polarity is a defining feature of all organisms. Until very recently, it was thought that delivery of proteins to polar domains of root epidermal cells plasma membrane was non-polar, but this view has been re-examined, the delivery is polar but the dynamics, the paths taken, and the mechanisms are unknown. My host team previously characterised an enrichment of Very-Long-Chain-Fatty-Acids (VLCFAs)-containing sphingolipids at the site of secretory vesicles (SVs) sub-domain of the trans-Golgi Network (TGN). Moreover, the length of sphingolipids acyl-chain was found to play a critical role in secretory sorting of the auxin carrier PIN2 from SVsassociated TGN to apical polar domain of the plasma membrane (PM). During my PhD, I addressed the following question: how sphingolipids act at SVs/TGN? Using proteomics of SVs, genetics and pharmacological tools in combination with visualisation of lipid probes we could identify that sphingolipids act on phosphoinositides (PIPs) homeostasis establishing a new functional link between these two lipids in plant cells. Using a set of multi-affinity fluorescent PIPs probes I could show that sphingolipids target phosphatidylinositol-3-phosphate (PI3P) and phosphatidylinositol-4-phosphate (PI4P). Moreover, my proteomic analyses show that several PIPs-related proteins are downregulated in immuno-purified TGN-associated SVs when the sphingolipid composition is altered pharmacologically. My results force the reassessment of our view of lipid membranes dynamics and highlight the idea that dynamic remodelling of the composition of one lipid class, the phosphoinositides, can be modulated by another lipid class, the sphingolipids., La polarité cellulaire est une caractéristique commune à tous les organismes. Jusqu’à récemment, il était assumé que la sécrétion de protéines vers des domaines polaires de la cellule végétale se faisait de façon non polarisée, mais ce point de vue a été re-étudié, la sécrétion est polarisée mais la dynamique, les voies de traficempruntées et les mécanismes sont toujours inconnus. Précédemment, mon laboratoire d’accueil a caractérisé un enrichissement en sphingolipides contenant des acides gras à très longues chaines (VLCFAs) au niveau d’un sous-domaine du trans-Golgi Network (TGN) appelé Vésicules de Sécrétions (SVs). Plus précisément, il a été montré que la longueur des acides gras des sphingolipides jouait un rôle critique dans la sécrétion du transporteur d’auxine PIN2 des SVs vers des domaines polaires de la membrane plasmique. Pendant ma thèse, je me suis intéressé à la question suivante : comment les sphingolipides agissent-t-ils au TGN? En identifiant le protéome des SVs, ainsi qu'en utilisant des outils génétiques et pharmacologiques en combinaison avec la visualisation de marqueurs lipidiques, j'ai pu identifier que les sphingolipides agissent sur l’homéostasie des phosphoinositides en mettant en avant un lien fonctionnel entre ces deux classes de lipides au sein de la cellule végétale. En utilisant un set de marqueurs des phosphoinositides (PIPs), j’ai pu montrer que les sphingolipides ciblent principalement le phosphatidyl-inositol-3-phosphate, PI(3)P et le phosphatidylinositol- 4-phosphate, PI(4)P. De plus, mon analyse protéomique a montré que la localisation d'un ensemble de protéines liées aux PIPs était diminuée dans les SVs/TGN immunopurifiées quand la composition des sphingolipides est altérée. Mes résultats nous forcent à revoir notre vision de la dynamique des lipides au niveau des membranes, et suggère l’idée que la dynamique de remodelage de la composition d’une classe de lipide, les phosphoinositides, peut être modulée par une autre classe de lipide, les sphingolipides.

The post-Golgi compartment trans-Golgi Network (TGN) is a central hub divided into multiple subdomains hosting distinct trafficking pathways, including polar delivery to apical membrane. Lipids such as sphingolipids and sterols have been implicated in polar trafficking from the TGN but the underlying mechanisms linking lipid composition to functional polar sorting at TGN subdomains remain unknown. Here we demonstrate that sphingolipids with α-hydroxylated acyl-chains of at least 24 carbon atoms are enriched in secretory vesicle subdomains of the TGN and are critical for de novo polar secretory sorting of the auxin carrier PIN2 to apical membrane of Arabidopsis root epithelial cells. We show that sphingolipid acyl-chain length influences the morphology and interconnections of TGN-associated secretory vesicles. Our results uncover that the sphingolipids acyl-chain length links lipid composition of TGN subdomains with polar secretory trafficking of PIN2 to apical membrane of polarized epithelial cells., Sphingolipids in the trans-Golgi network have been implicated in polar trafficking. Here Wattelet-Boyer et al. show that hydroxylated C24- and C26-acyl-chain sphingolipids are enriched in trans-Golgi network subdomains that are critical for polar sorting of the PIN2 auxin carrier in plant cells.