Aquaporines intracellulaires d'Arabidopsis thaliana : dynamique d'expression dans le pollen et dans la racine sous stress oxydatif

Les aquaporines sont des canaux hydriques qui contrôlent la perméabilité à l'eau des membranes cellulaires, au cours du développement ou en réponse à des stress. La dynamique de l'expression des aquaporines de plantes et leur rôle physiologique ont été examinés dans deux organes modèles, le pollen et la racine d'Arabidopsis. Le pollen mature contient une cellule végétative et deux cellules de sperme. Des analyses transcriptomiques ont récemment identifié AtTIP1;3 et AtTIP5;1 comme deux aquaporines spécifiques du pollen. Dans ce travail, des protéines reportrices fluorescentes ont permis d'établir que AtTIP1;3 et AtTIP5;1 s'expriment spécifiquement dans la membrane vacuolaire de, respectivement, la cellule végétative et les cellules de sperme. Ces études révèlent aussi la grande plasticité dynamique des vacuoles, de la maturation du pollen jusqu'à la fécondation. Des approches de génétique inverse suggèrent un rôle des deux aquaporines dans la reproduction de la plante. La seconde partie de ce travail concerne les effets concomitants des stress oxydants, inhibant la conductivité hydraulique des racines et provoquant une accumulation intracellulaire des aquaporines initialement sur les membranes plasmiques. Le dernier processus a été disséqué par des approches de biochimie, pharmacologie et microscopie. La co-expression avec des marqueurs des endomembranes a révélé que l'isoforme AtPIP2;1 subit une accumulation dans les endosomes tardifs en réponse à l'H2O2. Ce processus peut être bloqué par l'auxine synthétique 1-NAA, mais non par l'inhibiteur d'endocytose tyrphostine A23. La grande stabilité des aquaporines internalisées suggère que l'H2O2 déclenche un mécanisme de séquestration réversible de celles-ci. Au-delà de données originales sur la régulation cellulaire des aquaporines, ce travail apporte un éclairage nouveau sur la dynamique des membranes intracellulaires des plantes, au cours du développement ou en réponse à des stressAquaporins are membrane water channel proteins that mediate the fine-tuning of cell membrane water permeability during development or in response to environmental stresses. The dynamic expression of aquaporins in planta, as well as their role in plant water relations, were investigated in two representative model organs, the pollen and roots of Arabidopsis. Mature pollen consists of a vegetative cell and two sperm cells. Transcriptomics recently identified AtTIP1;3 and AtTIP5;1 as two pollen exclusive aquaporins. Here, we investigated their in vivo temporal and spatial expression pattern. Fluorescently-tagged chimeras revealed that AtTIP1;3 and AtTIP5;1 have a distinct and specific localisation in the vacuolar membrane of the vegetative and sperm cells, respectively. The two aquaporins also revealed the dynamic plasticity of vacuoles from pollen maturation to embryo fecundation. Loss of function approaches suggest an implication of both proteins in plant reproduction. The second part of this work focused on the oxidative stress-induced internalisation of root plasma membrane aquaporins and its concomitant drop in root hydraulic conductivity. The former process was described in great detail by combined biochemical, pharmacological and microscopic approaches. Co-expression analyses of the AtPIP2;1 isoform with endomembrane markers revealed that H2O2 triggers AtPIP2;1 accumulation in late endosomal compartments. This process could be antagonized by the auxin analog 1-NAA, but not by the endocytosis blocker tyrphostin A23. Life-time analyses established the high stability of the internalised protein suggesting that H2O2 triggers a mechanism for intracellular and reversible sequestration of plasma membrane aquaporins. Besides information on cell regulation of aquaporins, the overall work gives novel and complementary insights into the dynamic remodelling of plant internal membranes during development and stress responses

Signaling with Ions: The Keystone for Apical Cell Growth and Morphogenesis in Pollen Tubes

info:eu-repo/semantics/publishedVersio

A look inside: localization patterns and functions of intracellular plant aquaporins.

International audienceAquaporins form a superfamily of intrinsic channel proteins in the plasma and intracellular membranes of plant cells. While a lot of research effort has substantiated the importance of plasma membrane aquaporins for the regulation of plant water homeostasis, comparably little is known about the function of intracellular aquaporins. Yet, various low-molecular-weight compounds, in addition to water, were recently shown to permeate some of these aquaporins. In this review, we examine the diversity of transport properties and localization patterns of intracellular aquaporins. The discussed profiles include, for example, water and ammonia transport across the tonoplast or CO2 transport through the chloroplast envelope. Furthermore, we try to assess to what extent the diverse aquaporin distribution patterns, in relation to the high degree of compartmentation of plant cells, can be linked to a wide range of cellular functions

The cellular dynamics of plant aquaporin expression and functions.

International audienceAquaporins are channel proteins that facilitate the transport of water and small neutral molecules, including gases, across cell membranes of most of the living organisms. Integrative studies have stressed the role of aquaporins in maintaining the whole plant water and nutrient status. Cellular aspects of plant aquaporin functions and regulations are also extensively investigated. The present review provides a glance at recent progresses in this area. One first direction concerns the mechanisms that determine aquaporin targeting to specific subcellular membranes and a dynamic and stimulus-dependent control of their density in these membranes. The regulation of aquaporin opening and closing and its links to cell signalling cascades are also discussed. Multiple cellular functions are now attributed to plant aquaporins. They include the dynamic equilibration and subcellular partitioning of their various substrates and a contribution to cell expansion and possibly cell division

Vegetative and sperm cell-specific aquaporins of Arabidopsis thaliana highlight the vacuolar equipment of pollen and contribute to plant reproduction.

International audience: The water and nutrient status of pollen is crucial to plant reproduction. Pollen grains of Arabidopsis thaliana contain a large vegetative cell and two smaller sperm cells. Pollen grains express AtTIP1;3 and AtTIP5;1, two members of the Tonoplast Intrinsic Protein sub-family of aquaporins. To address the spatial and temporal expression pattern of the two homologues, C-terminal fusions of AtTIP1;3 and AtTIP5;1 with GFP and mCherry, respectively, were expressed in transgenic Arabidopsis under the control of their native promoter. Confocal laser scanning microscopy revealed that AtTIP1;3 and AtTIP5;1 are specific for the vacuoles of the vegetative and sperm cells, respectively. The tonoplast localization of AtTIP5;1 was established by reference to fluorescent protein markers for the mitochondria and vacuoles of sperm and vegetative cells and is at variance with a recent work (Soto et al., 2010, Plant J 64: 1038-1047) which localized AtTIP5;1 in vegetative cell mitochondria. AtTIP1;3-GFP and AtTIP5;1-mCherry showed concomitant expression, from first pollen mitosis up to pollen tube penetration in the ovule, thereby revealing the dynamics of vacuole morphology in maturating and germinating pollen. T-DNA insertion mutants for either AtTIP1;3 or AtTIP5;1 showed no apparent growth phenotype and had no significant defect in male transmission of the mutated alleles. By contrast, a double knock-out displayed an abnormal rate of barren siliques, this phenotype being more pronounced under limiting water or nutrient supply. The overall data indicate that vacuoles of vegetative and sperm cells functionally interact and contribute to male fertility in adverse environmental conditions

Aquaporins and plant adaptation to a changing environment

Aquaporins and plant adaptation to a changing environment. Environmental Workshop of Universidad Internacional de Andalucia: Genomic, physiological and breeding approaches for enhancing drought resistance in crop

Affinity Purification of GO-Matryoshka Biosensors from E. coli for Quantitative Ratiometric Fluorescence Analyses

Genetically encoded biosensors are powerful tools for quantitative visualization of ions and metabolites in vivo. Design and optimization of such biosensors typically require analyses of large numbers of variants. Sensor properties determined in vitro such as substrate specificity, affinity, response range, dynamic range, and signal-to-noise ratio are important for evaluating in vivo data. This protocol provides a robust methodology for in vitro binding assays of newly designed sensors. Here we present a detailed protocol for purification and in vitro characterization of genetically encoded sensors, exemplified for the His affinity-tagged GO-(Green-Orange) MatryoshCaMP6s calcium sensor. GO-Matryoshka sensors are based on single-step insertion of a cassette containing two nested fluorescent proteins, circularly permutated fluorescent green FP (cpGFP) and Large Stoke Shift LSSmOrange, within the binding protein of interest, producing ratiometric sensors that exploit the analyte-triggered change in fluorescence of a cpGFP

Plant aquaporin endomembrane trafficking and dynamics

Plant aquaporin endomembrane trafficking and dynamics. 13th International Workshop on Plant Membrane Biolog

Assembly and Sorting of the Tonoplast Potassium Channel AtTPK1 and Its Turnover by Internalization into the Vacuole1[W][OA]

The assembly, sorting signals, and turnover of the tonoplast potassium channel AtTPK1 of Arabidopsis (Arabidopsis thaliana) were studied. We used transgenic Arabidopsis expressing a TPK1-green fluorescent protein (GFP) fusion or protoplasts transiently transformed with chimeric constructs based on domain exchange between TPK1 and TPK4, the only TPK family member not located at the tonoplast. The results show that TPK1-GFP is a dimer and that the newly synthesized polypeptides transiently interact with a thus-far unidentified 20-kD polypeptide. A subset of the TPK1-TPK4 chimeras were unable to assemble correctly and these remained located in the endoplasmic reticulum where they interacted with the binding protein chaperone. Therefore, TPK1 must assemble correctly to pass endoplasmic reticulum quality control. Substitution of the cytosolic C terminus of TPK4 with the corresponding domain of TPK1 was sufficient to allow tonoplast delivery, indicating that this domain contains tonoplast sorting information. Pulse-chase labeling indicated that TPK1-GFP has a half-life of at least 24 h. Turnover of the fusion protein involves internalization into the vacuole where the GFP domain is released. This indicates a possible mechanism for the turnover of tonoplast proteins