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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.


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Decay in IK,inand IK,outfrom guard cells of wild-type Arabidopsis plants without (no pretreatment) and with opening buffer pretreatment (pretreatment)Decay in IK,inand IK,outfrom guard cells of wild-type Arabidopsis plants without (no pretreatment) and with opening buffer pretreatment (pretreatment). Voltage clamp scans were carried out at intervals following impalements. Raw current traces are shown in for scans at 10, 20, and 30 min time points from two guard cells for IK,out (A) and IK,in (C). Scale: vertical, 500 μA cm-2; horizontal, 2 s. Clamp scans were from a holding voltage of −100 mV with tail steps to −100 mV. Test voltage steps were to voltages between −80 and +50 mV for IK,out and to voltages between −100 and −240 mV for IK,in. Data in (B) summarise the two current amplitude means ± SE (filled circles, no pretreatment; open circles, pretreatment) from 12 independent experiments with IK,out determined at +40 mV and IK,in determined at −220 mV. Note that currents recorded from guard cells in control experiments without OB pretreatment generally decayed with halftimes of 15–20 min.
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Figure 4: Decay in IK,inand IK,outfrom guard cells of wild-type Arabidopsis plants without (no pretreatment) and with opening buffer pretreatment (pretreatment)Decay in IK,inand IK,outfrom guard cells of wild-type Arabidopsis plants without (no pretreatment) and with opening buffer pretreatment (pretreatment). Voltage clamp scans were carried out at intervals following impalements. Raw current traces are shown in for scans at 10, 20, and 30 min time points from two guard cells for IK,out (A) and IK,in (C). Scale: vertical, 500 μA cm-2; horizontal, 2 s. Clamp scans were from a holding voltage of −100 mV with tail steps to −100 mV. Test voltage steps were to voltages between −80 and +50 mV for IK,out and to voltages between −100 and −240 mV for IK,in. Data in (B) summarise the two current amplitude means ± SE (filled circles, no pretreatment; open circles, pretreatment) from 12 independent experiments with IK,out determined at +40 mV and IK,in determined at −220 mV. Note that currents recorded from guard cells in control experiments without OB pretreatment generally decayed with halftimes of 15–20 min.

Mentions: Out of 275 independent experiments with measurements of the K+ currents 88% showed IK,in activity and 100% yielded IK,out activity as judged by the current activation kinetics, voltage dependencies and block by Cs+ and TEA+ (not shown, see Roelfsema and Prins [26,27], Forestier et al. [28] and Blatt et al. [38]). Guard cells pretreated with OB showed appreciably greater stability in both IK,in and IK,out over extended time periods compared with guard cells impaled without pretreatment (Figures 3, 4 and Tables 3 and 4). Mean IK,in and IK,out amplitudes of all of the lines tested at 30 min, for example, decayed to less than 2% and 22%, respectively, of the initial amplitudes recorded 10 min after impalements in guard cells without OB pretreatment (see also [26]). By contrast, the K+ currents showed less than a 5% change in amplitude over the same time period when guard cells were first pretreated in OB.


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

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

Decay in IK,inand IK,outfrom guard cells of wild-type Arabidopsis plants without (no pretreatment) and with opening buffer pretreatment (pretreatment)Decay in IK,inand IK,outfrom guard cells of wild-type Arabidopsis plants without (no pretreatment) and with opening buffer pretreatment (pretreatment). Voltage clamp scans were carried out at intervals following impalements. Raw current traces are shown in for scans at 10, 20, and 30 min time points from two guard cells for IK,out (A) and IK,in (C). Scale: vertical, 500 μA cm-2; horizontal, 2 s. Clamp scans were from a holding voltage of −100 mV with tail steps to −100 mV. Test voltage steps were to voltages between −80 and +50 mV for IK,out and to voltages between −100 and −240 mV for IK,in. Data in (B) summarise the two current amplitude means ± SE (filled circles, no pretreatment; open circles, pretreatment) from 12 independent experiments with IK,out determined at +40 mV and IK,in determined at −220 mV. Note that currents recorded from guard cells in control experiments without OB pretreatment generally decayed with halftimes of 15–20 min.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Decay in IK,inand IK,outfrom guard cells of wild-type Arabidopsis plants without (no pretreatment) and with opening buffer pretreatment (pretreatment)Decay in IK,inand IK,outfrom guard cells of wild-type Arabidopsis plants without (no pretreatment) and with opening buffer pretreatment (pretreatment). Voltage clamp scans were carried out at intervals following impalements. Raw current traces are shown in for scans at 10, 20, and 30 min time points from two guard cells for IK,out (A) and IK,in (C). Scale: vertical, 500 μA cm-2; horizontal, 2 s. Clamp scans were from a holding voltage of −100 mV with tail steps to −100 mV. Test voltage steps were to voltages between −80 and +50 mV for IK,out and to voltages between −100 and −240 mV for IK,in. Data in (B) summarise the two current amplitude means ± SE (filled circles, no pretreatment; open circles, pretreatment) from 12 independent experiments with IK,out determined at +40 mV and IK,in determined at −220 mV. Note that currents recorded from guard cells in control experiments without OB pretreatment generally decayed with halftimes of 15–20 min.
Mentions: Out of 275 independent experiments with measurements of the K+ currents 88% showed IK,in activity and 100% yielded IK,out activity as judged by the current activation kinetics, voltage dependencies and block by Cs+ and TEA+ (not shown, see Roelfsema and Prins [26,27], Forestier et al. [28] and Blatt et al. [38]). Guard cells pretreated with OB showed appreciably greater stability in both IK,in and IK,out over extended time periods compared with guard cells impaled without pretreatment (Figures 3, 4 and Tables 3 and 4). Mean IK,in and IK,out amplitudes of all of the lines tested at 30 min, for example, decayed to less than 2% and 22%, respectively, of the initial amplitudes recorded 10 min after impalements in guard cells without OB pretreatment (see also [26]). By contrast, the K+ currents showed less than a 5% change in amplitude over the same time period when guard cells were first pretreated in OB.

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