A cell membrane can be considered a liquid-phase plane in which lipids and proteins theoretically are free to diffuse. Numerous reports, however, describe retarded diffusion of membrane proteins in animal cells. This anomalous diffusion results from a combination of structuring factors including protein-protein interactions, cytoskeleton corralling, and lipid organization into microdomains. In plant cells, plasma-membrane (PM) proteins have been described as relatively immobile, but the control mechanisms that structure the PM have not been studied. Here, we use fluorescence recovery after photobleaching to estimate mobility of a set of minimal PM proteins. These proteins consist only of a PM-anchoring domain fused to a fluorescent protein, but their mobilities remained limited, as is the case for many full-length proteins. Neither the cytoskeleton nor membrane microdomain structure was involved in constraining the diffusion of these proteins. The cell wall, however, was shown to have a crucial role in immobilizing PM proteins. In addition, by single-molecule fluorescence imaging we confirmed that the pattern of cellulose deposition in the cell wall affects the trajectory and speed of PM protein diffusion. Regulation of PM protein dynamics by the plant cell wall can be interpreted as a mechanism for regulating protein interactions in processes such as trafficking and signal transduction.

Rho guanosine triphosphatases (GTPases) are master regulators of cell signaling, but how they are regulated depending on the cellular context is unclear. We found that the phospholipid phosphatidylserine acts as a developmentally controlled lipid rheostat that tunes Rho GTPase signaling in Arabidopsis. Live superresolution single-molecule imaging revealed that the protein Rho of Plants 6 (ROP6) is stabilized by phosphatidylserine into plasma membrane nanodomains, which are required for auxin signaling. Our experiments also revealed that the plasma membrane phosphatidylserine content varies during plant root development and that the level of phosphatidylserine modulates the quantity of ROP6 nanoclusters induced by auxin and hence downstream signaling, including regulation of endocytosis and gravitropism. Our work shows that variations in phosphatidylserine levels are a physiological process that may be leveraged to regulate small GTPase signaling during development.

Membrane surface charge is critical for the transient, yet specific recruitment of proteins with polybasic regions to certain organelles. In eukaryotes, the plasma membrane (PM) is the most electronegative compartment of the cell, which specifies its identity. As such, membrane electrostatics is a central parameter in signaling, intracellular trafficking, and polarity. Here, we explore which are the lipids that control membrane electrostatics using plants as a model. We show that phosphatidylinositol-4-phosphate (PI4P), phosphatidic acidic (PA), and phosphatidylserine (PS) are separately required to generate the electrostatic signature of the plant PM. In addition, we reveal the existence of an electrostatic territory that is organized as a gradient along the endocytic pathway and is controlled by PS/PI4P combination. Altogether, we propose that combinatorial lipid composition of the cytosolic leaflet of organelles not only defines the electrostatic territory but also distinguishes different functional compartments within this territory by specifying their varying surface charges.

Cardiolipin (CL) is the signature phospholipid of the mitochondrial inner membrane. In animals and yeast (Saccharomyces cerevisiae), CL depletion affects the stability of respiratory supercomplexes and is thus crucial to the energy metabolism of obligate aerobes. In eukaryotes, the last step of CL synthesis is catalyzed by CARDIOLIPIN SYNTHASE (CLS), encoded by a single-copy gene. Here, we characterize a cls mutant in Arabidopsis thaliana, which is devoid of CL. In contrast to yeast cls, where development is little affected, Arabidopsis cls seedlings are slow developing under short-day conditions in vitro and die if they are transferred to long-day (LD) conditions. However, when transferred to soil under LD conditions under low light, cls plants can reach the flowering stage, but they are not fertile. The cls mitochondria display abnormal ultrastructure and reduced content of respiratory complex I/complex III supercomplexes. The marked accumulation of tricarboxylic acid cycle derivatives and amino acids demonstrates mitochondrial dysfunction. Mitochondrial and chloroplastic antioxidant transcripts are overexpressed in cls leaves, and cls protoplasts are more sensitive to programmed cell death effectors, UV light, and heat shock. Our results show that CLS is crucial for correct mitochondrial function and development in Arabidopsis under both optimal and stress conditions.

21Membrane surface charge is critical for the transient, yet specific recruitment of proteins with polybasic 22 regions to certain organelles. In all eukaryotes, the plasma membrane (PM) is the most electronegative 23 compartment of the cell, which specifies its identity. As such, membrane electrostatics is a central parameter 24 in signaling, intracellular trafficking and polarity. Here, we explore which are the lipids that control membrane 25 electrostatics using plants as a model. We show that phosphatidic acidic (PA), phosphatidylserine (PS) and 26 phosphatidylinositol-4-phosphate (PI4P) are separately required to generate the electrostatic signature of the 27 plant PM. In addition, we reveal the existence of an electrostatic territory that is organized as a gradient along 28 the endocytic pathway and is controlled by PS/PI4P combination. Altogether, we propose that combinatorial 29 lipid composition of the cytosolic leaflet of cellular organelles not only defines the plant electrostatic territory 30 but also distinguishes different compartments within this territory by specifying their varying surface charges.

Lipids have an established role as structural components of membranes or as signalling molecules, but their role as molecular actors in protein secretion is less clear. The complex sphingolipid glucosylceramide (GlcCer) is enriched in the plasma membrane and lipid microdomains of plant cells, but compared to animal and yeast cells, little is known about the role of GlcCer in plant physiology. We have investigated the influence of GlcCer biosynthesis by glucosylceramide synthase (GCS) on the efficiency of protein transport through the plant secretory pathway and on the maintenance of normal Golgi structure. We determined that GlcCer is synthesized at the beginning of the plant secretory pathway [mainly endoplasmic reticulum (ER)] and that D,L-threo-1-phenyl-2-decanoyl amino-3-morpholinopropanol (PDMP) is a potent inhibitor of plant GCS activity in vitro and in vivo. By an in vivo confocal microscopy approach in tobacco leaves infiltrated with PDMP, we showed that the decrease in GlcCer biosynthesis disturbed the transport of soluble and membrane secretory proteins to the cell surface, as these proteins were partly retained intracellularly in the ER and/or Golgi. Electron microscopic observations of Arabidopsis thaliana root cells after high-pressure freezing and freeze substitution evidenced strong morphological changes in the Golgi bodies, pointing to a link between decreased protein secretion and perturbations of Golgi structure following inhibition of GlcCer biosynthesis in plant cells.

The biosynthesis of most of the phospholipid species of the plasma membrane of plant cells, as in animal cells, takes place primarily in the ER (Moore, 1990). This is the case for PS for which an intracellular transport from the ER to the plasma membrane was postulated and demonstrated in vivo. This transport is inhibited by monensin and low temperatures and follows the ER-Golgi-plasma membrane pathway (Sturbois-Balcerzak et al., 1995; Moreau et al., 1998a). Therefore, this transport is expected to be mediated by carrier vesicles. Such structures can transport membrane and secretory proteins in plant cells (SatiatJeunemaitre and Hawes, 1993; Bar-Peled et al., 1996), and we also suspect their involvement in lipid transport (Moreau et al., 1988(Moreau et al., , 1998a Bertho et al., 1991; SturboisBalcerzak et al., 1995).Few attempts have concerned the isolation of putative vesicular intermediates involved in the delivery of membrane material from the ER in plant cells (Morré et al., 1989; Hellgren et al., 1993). Recently, a cell-free ATP-dependent transfer of phospholipids was obtained between the ER and the Golgi apparatus of leek (Allium porrum) cells (Sturbois et al., 1994). PC, PE, and particularly PS were transferred, mimicking the in vivo situation. On the other hand, PI was not found to be transported (Sturbois et al., 1994; Sturbois-Balcerzak et al., 1995).Although the characterization of proteins likely to be involved in vesicular transport is in progress in plant cells (Bar-Peled et al., 1996; Gomord and Faye, 1996; Hawes and Satiat-Jeunemaitre, 1996), there is still no specific marker for ER-derived vesicles in plant cells. We have developed another strategy to monitor the isolation of putative vesicular structures involved in the transport of phospholipids and especially PS in leek cells.It has been observed that ER-derived vesicles are 50-to 80-nm vesicular structures in many eukaryotic organisms (Paulik et al., 1988; Morré et al., 1989; Hellgren et al., 1993; Moreau et al., 1993; Bednarek et al., 1995). Moreover, the transport vesicles isolated from the ER of rat liver show an enrichment in PS (Moreau et al., 1992(Moreau et al., , 1993, and we have observed a selective transfer of PS in vivo (SturboisBalcerzak et al., 1995) and between the ER and the Golgi apparatus of leek cells in vitro (Sturbois et al., 1994).We incubated an ER-enriched membrane fraction from leek cells with ATP and other factors and observed the formation of small vesicles that were PS enriched. Their partial isolation was performed by sedimentation on Sucdensity gradients and/or filtration through 200-and 100-nm-pore membranes (Anotop, Anotec/Whatman). Our results show for the first time, to our knowledge, in a cell-free system from plant cells that phospholipids can be sorted and targeted from the ER to ER-derived vesicles, as is the case for proteins (Bar-Peled et al., 1996). 1 This work was supported by grants from the Centre National de la Recherche Scientifique and the University Victor Segalen Bordeaux 2. B.S.-B. w...

Anthocyanin biosynthesis is regulated by environmental factors (such as light, temperature, and water availability) and nutrient status (such as carbon, nitrogen, and phosphate nutrition). Previous reports show that low nitrogen availability strongly enhances anthocyanin accumulation in non carbon-limited plant organs or cell suspensions. It has been hypothesized that high carbon-to-nitrogen ratio would lead to an energy excess in plant cells, and that an increase in flavonoid pathway metabolic fluxes would act as an “energy escape valve,” helping plant cells to cope with energy and carbon excess. However, this hypothesis has never been tested directly. To this end, we used the grapevine Vitis vinifera L. cultivar Gamay Teinturier (syn. Gamay Freaux or Freaux Tintorier, VIVC #4382) cell suspension line as a model system to study the regulation of anthocyanin accumulation in response to nitrogen supply. The cells were sub-cultured in the presence of either control (25 mM) or low (5 mM) nitrate concentration. Targeted metabolomics and enzyme activity determinations were used to parametrize a constraint-based model describing both the central carbon and nitrogen metabolisms and the flavonoid (phenylpropanoid) pathway connected by the energy (ATP) and reducing power equivalents (NADPH and NADH) cofactors. The flux analysis (2 flux maps generated, for control and low nitrogen in culture medium) clearly showed that in low nitrogen-fed cells all the metabolic fluxes of central metabolism were decreased, whereas fluxes that consume energy and reducing power, were either increased (upper part of glycolysis, shikimate, and flavonoid pathway) or maintained (pentose phosphate pathway). Also, fluxes of flavanone 3β-hydroxylase, flavonol synthase, and anthocyanidin synthase were strongly increased, advocating for a regulation of the flavonoid pathway by alpha-ketoglutarate levels. These results strongly support the hypothesis of anthocyanin biosynthesis acting as an energy escape valve in plant cells, and they open new possibilities to manipulate flavonoid production in plant cells. They do not, however, support a role of anthocyanins as an effective mechanism for coping with carbon excess in high carbon to nitrogen ratio situations in grape cells. Instead, constraint-based modeling output and biomass analysis indicate that carbon excess is dealt with by vacuolar storage of soluble sugars.