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The second half of both “weeks” of the school is devoted to practical training workshops (3 to 5 persons per workshop). Each attendee will be asked to follow 2 workshops that she/he has to select amongst the 6 possibilities listed below

W1: Use of Ca²⁺ and ROS genetically encoded biosensors to unravel plant signaling in response to environment changes

Carine Alcon, Alexandre Martinière, Lionel Verdoucq, Tou Cheu Xiong (IPSiM, Montpellier)

Fluorescent biosensors are powerful tools to study plant responses to their environment. In this practical, we will use genetically encoded reporters to visualize intracellular changes of free Ca²⁺ and H₂O₂. With the newly developed HyPer7 H₂O₂-biosensor, we will explore the differences in the redox status between the plasma membrane and cytoplasm of cells. In addition, we will use the flg22 elicitor to follow the accumulation of H₂O₂ in the guard cell cytoplasm. In the second part of the practical, the free Ca²⁺ variation will be monitored with the fluorescent biosensor R-GECO-1 in intact A. thaliana leaves or seedlings to study signal propagation under mechanical or nutrient stresses. This will be carried out with epifluorescent macroscope and FLIM (fluorescence lifetime microscopy). The practical course will also include image analysis (Image J freeware) on the data acquired during the training.

W2: Voltage-clamp analyses of transporters and voltage-gated channels ("whole-cell" current recordings)

Martin Boeglin, Claire Corratgé-Faillie, Anne Aliénor Véry (IPSiM, Montpellier)

The voltage-clamp technique makes it possible to study in detail the functional properties of ion transport systems, such as ion selectivity, regulation of activity by membrane voltage, direction of transport... Attendees will record ion currents using the voltage-clamp technique on heterologously expressed transporters and ion channels in Xenopus oocytes. The data will be analyzed to extract functional properties.

W3: Local ion concentration and flux measurements at the surface of plant roots using ion-selective microelectrodes

Martin Boeglin, Claire Corratgé-Faillie, Anne Aliénor Véry (IPSiM, Montpellier)

Ion-selective microelectrodes allow localized ion concentration measurements, which can be applied to flux measurements at the surface of plant roots. Attendees will learn the principle of functioning of ion-selective electrodes, built their own electrodes and check their properties through calibrations in different ionic backgrounds, before performing measurements on plants and analyses.

W4: Initiation to proteomics and phosphoproteomics

Valérie Rofidal, Nathalie Berger, Vincent Demolombe, Christian Dubos (Mass Spectrometry platform, IPSiM, Montpellier)

This workshop will address the use of mass spectrometry to identify and quantify proteins in their native and phosphorylated forms. During the first day, an introduction to principles in mass spectrometry for protein identification and posttranslational modification identifcation will be provided. Then a proteomic experiment will be conducted including protein digestion, phosphopeptides enrichment and peptides analysis with the mass spectrometer. During the second day, raw data will be processed to identify and quantify proteins/peptides through the use of a pipeline of dedicated softwares. A critical analysis of the data will also be provided.

W5: Water transport across membranes, cells and organs. 

Amandine Crabos, Fabrice Bauget, Philippe Nacry, Lionel Verdoucq, Yann Boursiac (IPSiM Montpellier)

The aim of this practical is to exemplify water transport assays in various membrane, cell and organ systems. Water transport in purified membrane vesicles or wall-deprived cell systems (isolated plant protoplasts) will be monitored using various types of osmotic swelling or shrinking assays, including a technique developed in our lab. Measurement of whole root water transport will be performed using pressure chambers. In parallel, extraction of the root architecture and it dynamic will be obtained with the use of a phenotyping platform. Ultimately, all data will be gathered into a mathematical model for a comprehensive simulation of the root hydraulic architecture, from the membrane to the organ. All assays will be carried out in WT plants as well as putative contrasting genotypes.

W6: Yeast as a tool to study in vivo protein-protein interactions and functional complementation studies

Léon Dirick, Florence Vignols (IPSiM, Montpellier)

This training firstly aims at providing trainees with basic knowledge in yeast (Saccharomyces cerevisiae) as a living eukaryotic tool for functional assays. It also aims to provide trainees with methods to assess the involvement of nutrient transporters in protein-protein interaction networks, and/or their ability to complement the functions of yeast orthologs. To fulfill these objectives, Mistral students will be trained to both theory and methods to perform yeast-two hybrid screenings and yeast mutant complementation assays using Arabidopsis transporters.