Cell Biology

Plants recognize a wide variety of microbial molecules to detect and respond to potential invaders. Recognition of Microbe-Associated Molecular Patterns (MAMPs) by cell surface receptors initiate a cascade of biochemical responses that include, among others, ion fluxes across the plasma membrane. A consequence of such event is a decrease in the concentration of extracellular H⁺ ions, which can be experimentally detected in plant cell suspensions as a shift in the pH of the medium. Thus, similarly to reactive oxygen species (ROS) accumulation, phosphorylation of MAP kinases and induction of defense-related genes, MAMP-induced medium alkalinization can be used as a proxy for the activation of plant immune responses. Here, we describe a detailed protocol for the measurement of medium alkalinization of tobacco BY-2 cell suspensions upon treatment with two different MAMPs: chitohexamers derived from fungal cell walls (NAG6; N-acetylglucosamine) and the flagellin epitope flg22, found in the bacterial flagellum. This method provides a reliable and fast platform to access MAMP-Triggered Immunity (MTI) in tobacco cell suspensions and can be easily adapted to other plant species as well as to other MAMPs.

Rice plants release proton (H⁺) from root cells into rhizosphere area leading to the acidification of the rhizosphere and increased solubility of ferric iron complexes on the cell membrane, which is important for iron uptakes. Here, we present a detailed protocol to measure H⁺ flux in root hairs of transgenic rice seedlings and transgenic rice protoplasts by the Non-invasive Micro-test Technique (NMT). The NMT system is based on a non-invasive microelectrode technology that is automatically controlled by a computer, to achieve a three-dimensional, real-time, dynamic characterization of the concentration, velocity, and direction of a variety of molecules or ions. Because there is no need to directly contact the measured cells that could cause cell damage, we are able to obtain accurate and real time information on ion concentration. This is the first protocol that describes the non-invasive micro measurement technique of both root hairs and protoplasts in rice. In NMT, voltage differences are measured at two excursion points that are manipulated using a computer. Voltage differences can be converted into H⁺ fluxes using the ASET 2.0 (The imFlux^® software) and JCal v3.2.1 Software. Analysis of the H⁺ fluxes provides a simultaneous measure of the crossing of a localized region of the root surface in response to stress, which provides real-time in-situ detection of net ion transport across membranes. This method will promote use of NMT in plant biology.