Françoise Simon-Plas

Sugar transporters in plants and in their interactions with fungi

Sucrose and monosaccharide transporters mediate long distance transport of sugar from source to sink organs and constitute key components for carbon partitioning at the whole plant level and in interactions with fungi. Even if numerous families of plant sugar transporters are defined; efflux capacities, subcellular localization and association to membrane rafts have only been recently reported. On the fungal side, the investigation of sugar transport mechanisms in mutualistic and pathogenic interactions is now emerging. Here, we review the essential role of sugar transporters for distribution of carbohydrates inside plant cells, as well as for plant-fungal interaction functioning. Altogether these data highlight the need for a better comprehension of the mechanisms underlying sugar exchanges between fungi and their host plants.

Tobacco cells contain a protein, immunologically related to the neutrophil small G protein Rac2 and involved in elicitor-induced oxidative burst

© 1997 Federation of European Biochemical Societies.

Lipids of the Plant Plasma Membrane

The plasma membrane (PM) is arguably the most diverse membrane of the plant cell. Furthermore, the protein and lipid composition of the PM varies with cell type, developmental stage, and environment. Physical properties of lipids and associate proteins allow the formation of a barrier that is selectively permeable to macromolecules and solutes. As the plasma membrane delineates the interface between the cell and the environment, it is the primary part of signal recognition and transduction into intracellular responses for nutritional uptake/distribution, environmental responses, and developmental signaling. Many essential PM functions are carried out by proteinaceous components. However, PM lipids play a crucial role in determining cell structures regulating membrane fluidity and transducing signals. The composition and physical state of the lipid bilayer influence lipid–protein and protein–protein associations, membrane-bound enzyme activities, and transport capacity of membranes. Analyses of membrane function require highly selective and efficient purification methods. In this chapter, we first briefly review the methods to isolate PM from plant tissue and describe the lipid content of purified membranes. We further examine the involvement of different lipid species on signaling events that allow the plant cell to cope with environmental fluctuations. Finally, we discuss how regulated segregation of lipids inside the PM is of crucial importance to understand signaling mechanisms.

Constitutive expression of clathrin hub hinders elicitor-induced clathrin-mediated endocytosis and defense gene expression in plant cells

Endocytosis has been recently implicated in the signaling network associated with the recognition of microbes by plants. In a previous study, we showed that the elicitor cryptogein was able to induce clathrin‐mediated endocytosis (CME) in tobacco suspension cells. Herein, we investigate further the induced CME by means of a GFP‐tagged clathrin light chain and a CME inhibitor, the hub domain of clathrin heavy chain. Hub constitutive expression does affect neither cell growth nor constitutive endocytosis but abolishes cryptogein‐induced CME. Such an inhibition has no impact on early events in the cryptogein signaling pathway but reduces the expression of defense‐associated genes.

Plasma membrane sterol complexation, generated by filipin, triggers signaling responses in tobacco cells.

The effects of changes in plasma membrane (PM) sterol lateral organization and availability on the control of signaling pathways have been reported in various animal systems, but rarely assessed in plant cells. In the present study, the pentaene macrolide antibiotic filipin III, commonly used in animal systems as a sterol sequestrating agent, was applied to tobacco cells. We show that filipin can be used at a non-lethal concentration that still allows an homogeneous labeling of the plasma membrane and the formation of filipin-sterol complexes at the ultrastructural level. This filipin concentration triggers a rapid and transient NADPH oxidase-dependent production of reactive oxygen species, together with an increase in both medium alkalinization and conductivity. Pharmacological inhibition studies suggest that these signaling events may be regulated by phosphorylations and free calcium. By conducting FRAP experiments using the di-4-ANEPPDHQ probe and spectrofluorimetry using the Laurdan probe, we provide evidence for a filipin-induced increase in PM viscosity that is also regulated by phosphorylations. We conclude that filipin triggers ligand-independent signaling responses in plant cells. The present findings strongly suggest that changes in PM sterol availability could act as a sensor of the modifications of cell environment in plants leading to adaptive cell responses through regulated signaling processes.

Behavior of plant plasma membranes under hydrostatic pressure as monitored by fluorescent environment-sensitive probes.

We monitored the behavior of plasma membrane (PM) isolated from tobacco cells (BY-2) under hydrostatic pressures up to 3.5kbar at 30 degrees C, by steady-state fluorescence spectroscopy using the newly introduced environment-sensitive probe F2N12S and also Laurdan and di-4-ANEPPDHQ. The consequences of sterol depletion by methyl-beta-cyclodextrin were also studied. We found that application of hydrostatic pressure led to a marked decrease of hydration as probed by F2N12S and to an increase of the generalized polarization excitation (GPex) of Laurdan. We observed that the hydration effect of sterol depletion was maximal between 1 and 1.5 kbar but was much less important at higher pressures (above 2 kbar) where both parameters reached a plateau value. The presence of a highly dehydrated gel state, insensitive to the sterol content, was thus proposed above 2.5 kbar. However, the F2N12S polarity parameter and the di-4-ANEPPDHQ intensity ratio showed strong effect on sterol depletion, even at very high pressures (2.5-3.5 kbar), and supported the ability of sterols to modify the electrostatic properties of membrane, notably its dipole potential, in a highly dehydrated gel phase. We thus suggested that BY-2 PM undergoes a complex phase behavior in response to the hydrostatic pressure and we also emphasized the role of phytosterols to regulate the effects of high hydrostatic pressure on plant PM.

Cercospora beticola toxins. Use of fluorescent cyanine dye to study their effects on tobacco cell suspensions

Abstract The fluorescent dye 3,3′-diethylthiadicarbocyanine iodide [diS-C 2 -(5)] was used to observe plasmalemma transmembrane potential variations of tobacco cells treated with uncoupler (FCCP), respiratory inhibitors (azide and cyanide), and H + -ATPase inhibitors (DCCD and a carbanilate derivative). These chemicals induced an increase in fluorescence, indicating a dissipation of the transmembrane potential. The [diS-C 2 -(5)] was also used to study the effects of two Cercospora beticola toxins on tobacco cells. Changes in fluorescence of [diS-C 2 -(5)] suggested that these two toxins caused a dissipation of the transmembrane potential with a different magnitude whereas kinetics of their association with membranes were comparable.

Direct purification of detergent-insoluble membranes from Medicago truncatula root microsomes: comparison between floatation and sedimentation

BackgroundMembrane microdomains are defined as highly dynamic, sterol- and sphingolipid-enriched domains that resist to solubilization by non-ionic detergents. In plants, these so-called Detergent Insoluble Membrane (DIM) fractions have been isolated from plasma membrane by using conventional ultracentrifugation on density gradient (G). In animals, a rapid (R) protocol, based on sedimentation at low speed, which avoids the time-consuming sucrose gradient, has also been developed to recover DIMs from microsomes as starting material. In the current study, we sought to compare the ability of the Rapid protocol versus the Gradient one for isolating DIMs directly from microsomes of M. truncatula roots. For that purpose, Triton X-100 detergent-insoluble fractions recovered with the two methods were analyzed and compared for their sterol/sphingolipid content and proteome profiles.ResultsInferred from sterol enrichment, presence of typical sphingolipid long-chain bases from plants and canonical DIM protein markers, the possibility to prepare DIMs from M. truncatula root microsomes was confirmed both for the Rapid and Gradient protocols. Contrary to sphingolipids, the sterol and protein profiles of DIMs were found to depend on the method used. Namely, DIM fractions were differentially enriched in spinasterol and only shared 39% of common proteins as assessed by GeLC-MS/MS profiling. Quantitative analysis of protein indicated that each purification procedure generated a specific subset of DIM-enriched proteins from Medicago root microsomes. Remarkably, these two proteomes were found to display specific cellular localizations and biological functions. In silico analysis of membrane-associative features within R- and G-enriched proteins, relative to microsomes, showed that the most noticeable difference between the two proteomes corresponded to an increase in the proportion of predicted signal peptide-containing proteins after sedimentation (R) compared to its decrease after floatation (G), suggesting that secreted proteins likely contribute to the specificity of the R-DIM proteome.ConclusionsEven though microsomes were used as initial material, we showed that the protein composition of the G-DIM fraction still mostly mirrored that of plasmalemma-originating DIMs conventionally retrieved by floatation. In parallel, the possibility to isolate by low speed sedimentation DIM fractions that seem to target the late secretory pathway supports the existence of plant microdomains in other organelles.

Cercospora beticola toxins. Part XVII. The role of the beticolin/Mg2+ complexes in their biological activity Study of plasma membrane H+-ATPase, vacuolar H+-PPase, alkaline and acid phosphatases

Beticolin-1 and beticolin-2, yellow toxins produced by the phytopathogenic fungus Cercospora beticola, inhibit the plasma membrane H(+)-ATPase. Firstly, since beticolins are able to form complexes with Mg2+, the role of the beticolin/Mg2+ complexes in the inhibition of the plasma membrane proton pump has been investigated. Calculations indicate that beticolins could exist under several forms, in the H(+)-ATPase assay mixture, both free or complexed with Mg2+. However, the percentage inhibition of the H(+)-ATPase activity is correlated to the concentration of one single form of beticolin, the dimeric neutral complex Mg2H2B2, which appears to be the active form involved in the H(+)-ATPase inhibition. Secondly, since previous data suggested that beticolins could also be active against other Mg2(+)-dependent enzymes, we tested beticolin-1 on the vacuolar H(+)-PPase, which requires Mg2+ as co-substrate, and on the alkaline and acid phosphatases, which do not use Mg2+ as co-substrate. Only vacuolar H(+)-PPase is sensitive to beticolin-1, which suggests that beticolins are specific to enzymes that use a complex of Mg2+ as the substrate. The same Mg2H2B2 complex which is responsible of the plasma membrane H(+)-ATPase inhibition appears to be also involved in the inhibition of the vacuolar H(+)-PPase.

Regulation of Plant Transporters by Lipids and Microdomains

Transporters in the broad sense, that is, carriers, pumps, and channels, are proteins inserted in a lipid bilayer separating two cellular compartments. This lipid bilayer is not only the physical support of such proteins, but also a powerful way to regulate their activity. This chapter will first summarize the different means by which lipids can regulate the activity of transmembrane proteins (including the physical properties of the bilayer, its dynamic lateral compartmentalization, and the presence of particular lipid species acting as cofactors). It will then illustrate these general rules with examples of such regulations found in plant literature and, as a reference, in animal studies.