New insights into the tonoplast architecture of plant vacuoles and vacuolar dynamics during osmotic stress

BACKGROUND: The vegetative plant vacuole occupies >90% of the volume in mature plant cells. Vacuoles play fundamental roles in adjusting cellular homeostasis and allowing cell growth. The composition of the vacuole and the regulation of its volume depend on the coordinated activities of the transporters and channels localized in the membrane (named tonoplast) surrounding the vacuole. While the tonoplast protein complexes are well studied, the tonoplast itself is less well described. To extend our knowledge of how the vacuole folds inside the plant cell, we present three-dimensional reconstructions of vacuoles from tobacco suspension cells expressing the tonoplast aquaporin fusion gene BobTIP26-1::gfp. RESULTS: 3-D reconstruction of the cell vacuole made possible an accurate analysis of large spanning folds of the vacuolar membrane under both normal and stressed conditions, and suggested interactions between surrounding plastids. Dynamic, high resolution 3-D pictures of the vacuole in tobacco suspension cells monitored under different growth conditions provide additional details about vacuolar architecture. The GFP-decorated vacuole is a single continuous compartment transected by tubular-like transvacuolar strands and large membrane surfaces. Cell culture under osmotic stress led to a complex vacuolar network with an increased tonoplast surface area. In-depth 3-D realistic inspections showed that the unity of the vacuole is maintained during acclimation to osmotic stress. Vacuolar unity exhibited during stress adaptation, coupled with the intimate associations of vacuoles with other organelles, suggests a physiological role for the vacuole in metabolism, and communication between the vacuole and organelles, respectively, in plant cells. Desiccation stress ensuing from PEG treatment generates "double" membrane structures closely linked to the tonoplast within the vacuole. These membrane structures may serve as membrane reservoirs for membrane reversion when cells are reintroduced to normal growth conditions. CONCLUSION: 3-D processing of a GFP-labeled tonoplast provides compelling visual constructions of the plant cell vacuole and elaborates on the nature of tonoplast folding and architecture. Furthermore, these methods allow real-time determination of membrane rearrangements during stresses

Dynamique membranaire dans les réponses précoces des cellules de tabac à la cryptogéine

National audienc

Clathrin-mediated endocytosis: an early event triggered by an elicitor of defence reaction in tobacco

International audienc

Cryptogein treatment stimulates clathrin-endocytosis correlated with ROS production in tobacco cells

International audienceDuring plant pathogen interactions, specific molecules elicit defense response of plant cells. Cryptogein, an elicitin produced by the pathogenic fungus Phytophtora cryptogea, induces a hypersensitive response (HR) in tobacco plants. The mode of action for cryptogein starts with recognition of this elicitor by an unidentified plasma membrane (PM) receptor. This ligand-receptor binding trigger a cascade of events at the PM level including rapid calcium influx, ions effluxes, extracellular medium alcalinization and plasma membrane depolarization both in tobacco plants and suspension cells. These events lead to the activation of plasma membrane-bound NADPH oxidase, NtrbohD, that is responsive for reactive oxygen species (ROS) production (1). In animals cells, when extracellular ligands bind to their receptors at the plasma membrane, the resulting complexes are commonly internalized via receptor-mediated endocytosis (RME; Receptor Mediated Endocytosis). It has a much lower profile in plant systems, where the occurrence of endocytotic processes of receptors has been discovered recently, and where plasma membrane receptor-ligand interactions are incompletely characterized. But, a strong evidence for a specific ligand-stimulated uptake of receptors, component of plant innate immune response, was recently described in Arabidopsis (reviewed in 2). Using FM4-64 and confocal lasr microscopy, we demonstrated that the specific cryptogein-binding to the PM increases the endocytosis rate in tobacco BY-2 cells. By transmission electronic microscopy we have scrutinized PM dynamics during elicitation, and showed that clathrin-coated pit gathering was under the influence of cryptogein. A pharmalogical interference approach, recently used in Arabidopsis (3) confirms that a part of cryptogein-induced endocytosis concerns the clathrin-dependent pathway involved in receptor-mediated endocytosis. Moreover, a BY-2 cell line expressing NtrbohD antisens cDNA, which is enable to produce ROS when submitted to cryptogein (1), does not present cryptogein-stimulated endocytosis. These results suggest that the transitory ROS production triggered by cryptogein elicitation plays a role in the clathrin-endocytosis stimulation. (1) Simon-Plant et al. (2002). Plant Journal 31 (2), 137-147. (2) Robatzek et al. (2007). Cllular Microbiology 9 (1), 1-8. (3) Dhonukshe et al. (2007). Current Biology 17, 520-527

Clathrin-mediated endocytosis: an early event triggered by an elicitor of defence reaction in tobacco

International audienc

Membre du comité international d’organisation

International audienc

Membrane trafficking in immune singalin of plants

International audienceThroughout their life, plants face with a vast diversity of beneficial and pathogenicmicrobes. Following microbe recognition, whatever the outcome of the interaction,intensive changes in membrane vesicle trafficking to and from the plasma membraneoccur. This allows plant cells to modify the spatio-temporal distribution and/or theactivity of membrane proteins, influencing their molecular dialogue with microbes. Asexample, a secreted protein from the oomycete Phytophthora cryptogea elicitsdefense reactions against pathogens by a transcriptional reprogramming and celldeath of tobacco cells. In early steps, the microbe protein stimulates clathrin-mediated endocytosis and plasma membrane reorganization. These membraneprocesses are dependent on reactive oxygen species generated by the NADPHoxidase D. Interrelations between signaling events and membrane dynamics, whichcan ultimately trigger immune responses, will be discussed

Plasma Membrane Dynamics in Plant-Microbe Interactions

International audienc

Plasma membrane protein trafficking in plant-microbe interactions: a plant cell point of view

International audienceIn order to ensure their physiological and cellular functions, plasma membrane (PM) proteins must be properly conveyed from their site of synthesis, i.e., the endoplasmic reticulum, to their final destination, the PM, through the secretory pathway. PM protein homeostasis also relies on recycling and/or degradation, two processes that are initiated by endocytosis. Vesicular membrane trafficking events to and from the PM have been shown to be altered when plant cells are exposed to mutualistic or pathogenic microbes. In this review, we will describe the fine-tune regulation of such alterations, and their consequence in PM protein activity. We will consider the formation of intracellular perimicrobial compartments, the PM protein trafficking machinery of the host, and the delivery or retrieval of signaling and transport proteins such as pattern-recognition receptors, producers of reactive oxygen species, and sugar transporters

Regulation of endocytosis by external stimuli in plant cells

International audienceEndocytosis is an essential process for the normal growth and development of eukaryotic cells. This mini-review presents an overview of endocytosis regulation in challenging environmental conditions such as nutrient availability, hormonal treatment or abiotic/biotic stress in plant cells. Recent data establishing a relationship between endocytosis and reactive oxygen species (ROS) production are also discussed