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Protocol: optimised electrophyiological analysis of intact guard cells from Arabidopsis.

Chen ZH, Eisenach C, Xu XQ, Hills A, Blatt MR - Plant Methods (2012)

Bottom Line: Genetic resources available for Arabidopsis thaliana make this species particularly attractive as a model for molecular genetic studies of guard cell homeostasis, transport and signalling, but this facility is not matched by accessible tools for quantitative analysis of transport in the intact cell.We have developed a reliable set of procedures for voltage clamp analysis of guard cells from Arabidopsis leaves.These procedures greatly simplify electrophysiological recordings, extending the duration of measurements and scope for analysis of the predominant K+ and anion channels of intact stomatal guard cells to that achieved previously in work with Vicia and tobacco guard cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Plant Physiology and Biophysics, Institute of Molecular, Cell and Systems Biology, University of Glasgow, Glasgow, G12 8QQ, UK. Michael.Blatt@glasgow.ac.uk.

ABSTRACT
Genetic resources available for Arabidopsis thaliana make this species particularly attractive as a model for molecular genetic studies of guard cell homeostasis, transport and signalling, but this facility is not matched by accessible tools for quantitative analysis of transport in the intact cell. We have developed a reliable set of procedures for voltage clamp analysis of guard cells from Arabidopsis leaves. These procedures greatly simplify electrophysiological recordings, extending the duration of measurements and scope for analysis of the predominant K+ and anion channels of intact stomatal guard cells to that achieved previously in work with Vicia and tobacco guard cells.

No MeSH data available.


Related in: MedlinePlus

Mechanical improvements for Arabidopsis guard cell impalement.  (A) Double-barrelled microelectrodes pulled with settings for Vicia (above) and for Arabidopsis (below), in the latter case showing a 1–1.5o taper to the final 10 μm of the tip. The extreme tips of both microelectrodes are below the resolution of the light microscope. Scale bars, 10 μm. (B) A custom-built brace with a fixed clamp (fc) for one amplifier headstage and a second, adjustable clamp (ac) provided by a Narashige C2 micromanipulator. The entire brace is fixed to the lateral, rack-and-pinion coarse movement of a Huxley-type micromanipulator visible behind. Scale bar, 1 cm. (C) Halfcells of the Ag/AgCl-KCl type constructed (left) using 0.5 mm diameter Ag wire soldered to a 2-mm diameter socket threaded in a PTFE sleeve and fitted with silicon and glass tubing, and (right) using 0.5 mm diameter Ag wire soldered to a 2-mm diameter socket and press-fit with a silicon plug behind the tip segment of a 2-ml graduated polypropylene pipette tip. Scale bar, 1 cm. When backfilled with KCl electrolyte, the halfcells weigh 5.5 g (left) and 0.6 g (right). For general details of halfcell construction, see [9].
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Figure 2: Mechanical improvements for Arabidopsis guard cell impalement. (A) Double-barrelled microelectrodes pulled with settings for Vicia (above) and for Arabidopsis (below), in the latter case showing a 1–1.5o taper to the final 10 μm of the tip. The extreme tips of both microelectrodes are below the resolution of the light microscope. Scale bars, 10 μm. (B) A custom-built brace with a fixed clamp (fc) for one amplifier headstage and a second, adjustable clamp (ac) provided by a Narashige C2 micromanipulator. The entire brace is fixed to the lateral, rack-and-pinion coarse movement of a Huxley-type micromanipulator visible behind. Scale bar, 1 cm. (C) Halfcells of the Ag/AgCl-KCl type constructed (left) using 0.5 mm diameter Ag wire soldered to a 2-mm diameter socket threaded in a PTFE sleeve and fitted with silicon and glass tubing, and (right) using 0.5 mm diameter Ag wire soldered to a 2-mm diameter socket and press-fit with a silicon plug behind the tip segment of a 2-ml graduated polypropylene pipette tip. Scale bar, 1 cm. When backfilled with KCl electrolyte, the halfcells weigh 5.5 g (left) and 0.6 g (right). For general details of halfcell construction, see [9].

Mentions: 2. For double-barrelled microelectrodes with the higher input resistances (and correspondingly lower electrolyte leakage rates), pull double-barrelled microelectrodes, after twisting 360o[9], using settings to give a pull time around 25 s. NOTE: We use settings similar to those used for Vicia and tobacco guard cells[34], but with the coil heat elevated to give pull times roughly 25% less than used for Vicia guard cells. The resulting microelectrodes have 1.8-2.0 cm-long shanks and tips that tapered with a 1–1.5oangle (Figure2A).


Protocol: optimised electrophyiological analysis of intact guard cells from Arabidopsis.

Chen ZH, Eisenach C, Xu XQ, Hills A, Blatt MR - Plant Methods (2012)

Mechanical improvements for Arabidopsis guard cell impalement.  (A) Double-barrelled microelectrodes pulled with settings for Vicia (above) and for Arabidopsis (below), in the latter case showing a 1–1.5o taper to the final 10 μm of the tip. The extreme tips of both microelectrodes are below the resolution of the light microscope. Scale bars, 10 μm. (B) A custom-built brace with a fixed clamp (fc) for one amplifier headstage and a second, adjustable clamp (ac) provided by a Narashige C2 micromanipulator. The entire brace is fixed to the lateral, rack-and-pinion coarse movement of a Huxley-type micromanipulator visible behind. Scale bar, 1 cm. (C) Halfcells of the Ag/AgCl-KCl type constructed (left) using 0.5 mm diameter Ag wire soldered to a 2-mm diameter socket threaded in a PTFE sleeve and fitted with silicon and glass tubing, and (right) using 0.5 mm diameter Ag wire soldered to a 2-mm diameter socket and press-fit with a silicon plug behind the tip segment of a 2-ml graduated polypropylene pipette tip. Scale bar, 1 cm. When backfilled with KCl electrolyte, the halfcells weigh 5.5 g (left) and 0.6 g (right). For general details of halfcell construction, see [9].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475070&req=5

Figure 2: Mechanical improvements for Arabidopsis guard cell impalement. (A) Double-barrelled microelectrodes pulled with settings for Vicia (above) and for Arabidopsis (below), in the latter case showing a 1–1.5o taper to the final 10 μm of the tip. The extreme tips of both microelectrodes are below the resolution of the light microscope. Scale bars, 10 μm. (B) A custom-built brace with a fixed clamp (fc) for one amplifier headstage and a second, adjustable clamp (ac) provided by a Narashige C2 micromanipulator. The entire brace is fixed to the lateral, rack-and-pinion coarse movement of a Huxley-type micromanipulator visible behind. Scale bar, 1 cm. (C) Halfcells of the Ag/AgCl-KCl type constructed (left) using 0.5 mm diameter Ag wire soldered to a 2-mm diameter socket threaded in a PTFE sleeve and fitted with silicon and glass tubing, and (right) using 0.5 mm diameter Ag wire soldered to a 2-mm diameter socket and press-fit with a silicon plug behind the tip segment of a 2-ml graduated polypropylene pipette tip. Scale bar, 1 cm. When backfilled with KCl electrolyte, the halfcells weigh 5.5 g (left) and 0.6 g (right). For general details of halfcell construction, see [9].
Mentions: 2. For double-barrelled microelectrodes with the higher input resistances (and correspondingly lower electrolyte leakage rates), pull double-barrelled microelectrodes, after twisting 360o[9], using settings to give a pull time around 25 s. NOTE: We use settings similar to those used for Vicia and tobacco guard cells[34], but with the coil heat elevated to give pull times roughly 25% less than used for Vicia guard cells. The resulting microelectrodes have 1.8-2.0 cm-long shanks and tips that tapered with a 1–1.5oangle (Figure2A).

Bottom Line: Genetic resources available for Arabidopsis thaliana make this species particularly attractive as a model for molecular genetic studies of guard cell homeostasis, transport and signalling, but this facility is not matched by accessible tools for quantitative analysis of transport in the intact cell.We have developed a reliable set of procedures for voltage clamp analysis of guard cells from Arabidopsis leaves.These procedures greatly simplify electrophysiological recordings, extending the duration of measurements and scope for analysis of the predominant K+ and anion channels of intact stomatal guard cells to that achieved previously in work with Vicia and tobacco guard cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Plant Physiology and Biophysics, Institute of Molecular, Cell and Systems Biology, University of Glasgow, Glasgow, G12 8QQ, UK. Michael.Blatt@glasgow.ac.uk.

ABSTRACT
Genetic resources available for Arabidopsis thaliana make this species particularly attractive as a model for molecular genetic studies of guard cell homeostasis, transport and signalling, but this facility is not matched by accessible tools for quantitative analysis of transport in the intact cell. We have developed a reliable set of procedures for voltage clamp analysis of guard cells from Arabidopsis leaves. These procedures greatly simplify electrophysiological recordings, extending the duration of measurements and scope for analysis of the predominant K+ and anion channels of intact stomatal guard cells to that achieved previously in work with Vicia and tobacco guard cells.

No MeSH data available.


Related in: MedlinePlus