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.

Guard cell hydrogen peroxide and nitric oxide mediate elevated CO2‐induced stomatal movement in tomato

“…Guard cells from tomato plants were isolated essentially as described in the protocol developed for Arabidopsis (Pandey et al ., ; Leonhardt et al ., ; Zhang et al ., ) with minor modifications. Briefly, fully expanded young leaves ( c .…”

“…The basic solution and the enzyme solution I and II used were same as that described in Zhang et al . () except that two transcription inhibitors, actinomycin D (33 mg l −1 ) and cordycepin (100 mg l −1 ), were used in all procedures of the isolation, including the first step of blending (Leonhardt et al ., ).…”

“…Guard cells from tomato plants were isolated essentially as described in the protocol developed for Arabidopsis (Pandey et al, 2002;Leonhardt et al, 2004;Zhang et al, 2008) with minor modifications. Briefly, fully expanded young leaves (c. 25-30 leaves) with the major veins removed were blended for 50 s in a blender in 100 ml of cool distilled water, then filtered through a 100 lm nylon mesh.…”

Heterotrimeric G-protein regulation of ROS signalling and calcium currents in Arabidopsis guard cells

“…gpa1-3 and gpa1-4 are T-DNA insertional mutants in the Col ecotypic background that have been used extensively in studies of heterotrimeric G-protein function in Arabidopsis Temple and Jones, 2007;Zhang et al, 2008a); these mutants are documented by Western analysis to be null mutants at the protein level (Pandey et al, 2006 Guard cell protoplasts and patch clamping Young leaves of plants grown as described above were used for guard cell protoplast isolation, which was performed as reported previously (Pandey et al, 2002;Zhang et al, 2008b). Patch clamp electrophysiology was performed using previously established techniques (Pei et al, 2000;Zhang and Fan, 2009), including the use of Ba 2+ as the substitute charge carrier for Ca 2+ (Murata et al, 2001).…”

THI1, a Thiamine Thiazole Synthase, Interacts with Ca2+-Dependent Protein Kinase CPK33 and Modulates the S-Type Anion Channels and Stomatal Closure in Arabidopsis

“…Arabidopsis guard cell protoplasts were isolated according to Zhang et al (Zhang et al, 2008) with minor modification. Briefly, the abaxial epidermis were peeled from five to 10 fully expanded young leaves of 4-week-old Arabidopsis plants and then blended in 500 mL of distilled water for 30 s. The peels were filtered through a 100-mm nylon mesh and put into 5 mL of enzyme solution I, which contained 0.7% cellulysin cellulase, 0.1% PVP 40, 0.25% BSA in 55% basic solution (5 mM MES, 0.5 mM CaCl 2 , 0.5 mM MgCl 2 , 0.5 mM ascorbic acid, 10 mM KH 2 PO 4 , 0.55 M sorbitol, pH 5.5).…”