Journal
MOLECULAR PLANT
Volume 9, Issue 3, Pages 471-480Publisher
CELL PRESS
DOI: 10.1016/j.molp.2016.02.004
Keywords
guard cell; cytosolic Ca2+; cytosolic volume; voltage clamp; current injection; ion channel
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Funding
- Deutsche Forschungsgemeinschaft [GK 1342, FOR964]
- Pathogate'' [RO 2381/6-1]
- Deutsche Forschungsgemeinschaft (Pathogate'') [HE 1640/34-1]
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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 Ca2+ 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 Ca2+ concentration. A complete loss of voltage-induced Ca2+ signals occurred when Ca2+-permeable plasma membrane channels were simultaneously blocked with La3+. This shows that two mechanisms cause hyperpolarization-induced elevation of the cytosolic Ca2+-concentration: (i) activation of voltage-dependent Ca2+-permeable channels, (ii) osmotically induced expansion of the cytosol, which leads to a release of Ca2+ from intracellular stores.
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