Isolation and Whole-Cell Patch Clamping of Arabidopsis Guard Cell Protoplasts

Abstract

INTRODUCTIONThe flux of ions across membranes via ion channels is vital to cellular responses to internal and external stimuli, and therefore to cellular survival in changing circumstances. Patch clamping is a powerful technique for ion channel investigation, because it enables measurement of both net ion fluxes across the entire surface area of a cell and ion currents flowing through a single open channel. However, unlike animal cells, plant cells are surrounded by cell walls that prevent the physical contact between the patch pipette and the plasma membrane necessary for the patch clamp technique. To demonstrate how patch clamping can be applied to plant physiology research, we describe a protocol used to record potassium ion (K(+)) channel currents in Arabidopsis guard cell protoplasts (a widely studied model cell type in plant biology). The protocol requires a two-step cellulase and pectinase digestion to isolate high quality Arabidopsis guard cell protoplasts (i.e., plant cells lacking their cell walls), preparation of suitable glass capillary microelectrodes, and formation of the whole-cell configuration with a gigaohm (GΩ) seal. We also describe the history of the protocol and list other types of plant cells from which successful patch clamp recordings have been obtained.