title: Current Injection Provokes Rapid Expansion of the Guard Cell Cytosolic Volume and Triggers Ca(2+) Signals.
abstract: High-resolution microscopy opens the door for detailed single-cell studies with fluorescent reporter dyes and proteins. We used a confocal spinning disc microscope to monitor fluorescent dyes and the fluorescent protein Venus in tobacco and Arabidopsis guard cells. Multi-barreled microelectrodes were used to inject dyes and apply voltage pulses, which provoke transient rises in the cytosolic Ca(2+) level. Voltage pulses also caused changes in the distribution of Lucifer Yellow and Venus, which pointed to a reversible increase of guard cell cytosolic volume. The dynamic cytosolic volume changes turned out to be provoked by current injection of ions. A reduction of the clamp current, by blocking K(+) uptake channels with Cs(+), strongly suppressed the cytosolic volume changes. Cs(+) not only inhibited the expansion of the cytosol, but also inhibited hyperpolarization-induced elevations of the cytosolic Ca(2+) concentration. A complete loss of voltage-induced Ca(2+) signals occurred when Ca(2+)-permeable plasma membrane channels were simultaneously blocked with La(3+). This shows that two mechanisms cause hyperpolarization-induced elevation of the cytosolic Ca(2+)-concentration: (i) activation of voltage-dependent Ca(2+)-permeable channels, (ii) osmotically induced expansion of the cytosol, which leads to a release of Ca(2+) from intracellular stores.
authors.affiliation: Molecular Plant Physiology and Biophysics, Julius-von-Sachs Institute for Biosciences, Biocenter, Würzburg University, Julius-von-Sachs-Platz 2, 97082 Würzburg, Germany.
authors.forename: Lena J
authors.initials: LJ
authors.lastname: Voss
authors.affiliation: Molecular Plant Physiology and Biophysics, Julius-von-Sachs Institute for Biosciences, Biocenter, Würzburg University, Julius-von-Sachs-Platz 2, 97082 Würzburg, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Hedrich
authors.affiliation: Molecular Plant Physiology and Biophysics, Julius-von-Sachs Institute for Biosciences, Biocenter, Würzburg University, Julius-von-Sachs-Platz 2, 97082 Würzburg, Germany. Electronic address: roelfsema@botanik.uni-wuerzburg.de.
authors.forename: M Rob G
authors.initials: MRG
authors.lastname: Roelfsema
mesh_terms: D017360:Arabidopsis / Q000166:cytology; D020013:Calcium Signaling; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D048429:Cell Size / Q000187:drug effects; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D007811:Lanthanum / Q000494:pharmacology; D008839:Microelectrodes; D018613:Microscopy, Confocal; D010539:Permeability / Q000187:drug effects; D014026:Nicotiana / Q000166:cytology
chemical_list: D007811:Lanthanum
keywords: current injection; cytosolic Ca(2+); cytosolic volume; guard cell; ion channel; voltage clamp