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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.

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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.


IK,inand IK,outof wild-type (A), nia1nia2 (B), and QC3 (C) mutant Arabidopsis guard cells following pretreatment with opening buffer (OB). (A) Steady-state current–voltage curves for IK,in and IK,out from one guard cell of wild-type Arabidopsis recorded at intervals over 30 min after 2-h OB pretreatment. Shown are data for voltage clamp scans taken at 10 (closed circles), 20 (open circles), and 30 min (closed triangles) after impalement. 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. Current–voltage curves were fitted jointly to a Boltzmann function (solid lines) and yielding values for gmax of 3.8 and 6.3 μS cm-2, V1/2, of −181 and +1 mV, and δ of 1.9 and 1.8 for IK,in and IK,out, respectively. Insets: Current traces for time points at 30 min. Scale: 500 μA cm-2 vertical, 2 s horizontal. (B) Steady-state current–voltage curves for IK,in and IK,out from one guard cell of nia1nia2 mutant Arabidopsis recorded at intervals over 30 min after 2-h OB pretreatment. Shown are data for voltage clamp scans taken at 10 (closed circles), 20 (open circles), and 30 min (closed triangles) after impalement. Clamp voltage scans as above. Current–voltage curves were fitted jointly to a Boltzmann function (solid lines) and yielding values for gmax of 0.9 and 6.1 μS cm-2, V1/2, of −178 and +5 mV, and δ, of 1.8 and 1.8 for IK,in and IK,out, respectively. (C) Steady-state current–voltage curves for IK,in and IK,out from one guard cell of nia1nia2 mutant Arabidopsis recorded at intervals over 60 min after 2-h OB pretreatment. Shown are data for voltage clamp scans taken at 10 (closed circles), 20 (open circles), and 30 (closed triangles) 40 (open triangles), 50 (closed squares) and 60 (open squares) min after impalement. Clamp voltage scans as above. Current–voltage curves were fitted jointly to a Boltzmann function (solid lines) and yielding values for gmax of 4.1 and 4.6 μS cm-2, V1/2, -182 and −7 mV, and δ, of 1.7 and 1.9 for IK,in and IK,out, respectively. NOTE: Data analysis and curve fittings were carried out using SigmaPlot 11 (Systat Software, Inc., USA) and are reported, where appropriate, as means ± SE of n observations. Where appropriate significance was determined using Students’ T-test. Gating characteristics for IK,inand IK,outwere determined by fitting steady-state current–voltage curves to Eqn.(1)using non-linear, least-squares minimisation and the Marquardt-Levenberg algorithm[39].
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Figure 3: IK,inand IK,outof wild-type (A), nia1nia2 (B), and QC3 (C) mutant Arabidopsis guard cells following pretreatment with opening buffer (OB). (A) Steady-state current–voltage curves for IK,in and IK,out from one guard cell of wild-type Arabidopsis recorded at intervals over 30 min after 2-h OB pretreatment. Shown are data for voltage clamp scans taken at 10 (closed circles), 20 (open circles), and 30 min (closed triangles) after impalement. 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. Current–voltage curves were fitted jointly to a Boltzmann function (solid lines) and yielding values for gmax of 3.8 and 6.3 μS cm-2, V1/2, of −181 and +1 mV, and δ of 1.9 and 1.8 for IK,in and IK,out, respectively. Insets: Current traces for time points at 30 min. Scale: 500 μA cm-2 vertical, 2 s horizontal. (B) Steady-state current–voltage curves for IK,in and IK,out from one guard cell of nia1nia2 mutant Arabidopsis recorded at intervals over 30 min after 2-h OB pretreatment. Shown are data for voltage clamp scans taken at 10 (closed circles), 20 (open circles), and 30 min (closed triangles) after impalement. Clamp voltage scans as above. Current–voltage curves were fitted jointly to a Boltzmann function (solid lines) and yielding values for gmax of 0.9 and 6.1 μS cm-2, V1/2, of −178 and +5 mV, and δ, of 1.8 and 1.8 for IK,in and IK,out, respectively. (C) Steady-state current–voltage curves for IK,in and IK,out from one guard cell of nia1nia2 mutant Arabidopsis recorded at intervals over 60 min after 2-h OB pretreatment. Shown are data for voltage clamp scans taken at 10 (closed circles), 20 (open circles), and 30 (closed triangles) 40 (open triangles), 50 (closed squares) and 60 (open squares) min after impalement. Clamp voltage scans as above. Current–voltage curves were fitted jointly to a Boltzmann function (solid lines) and yielding values for gmax of 4.1 and 4.6 μS cm-2, V1/2, -182 and −7 mV, and δ, of 1.7 and 1.9 for IK,in and IK,out, respectively. NOTE: Data analysis and curve fittings were carried out using SigmaPlot 11 (Systat Software, Inc., USA) and are reported, where appropriate, as means ± SE of n observations. Where appropriate significance was determined using Students’ T-test. Gating characteristics for IK,inand IK,outwere determined by fitting steady-state current–voltage curves to Eqn.(1)using non-linear, least-squares minimisation and the Marquardt-Levenberg algorithm[39].

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)

IK,inand IK,outof wild-type (A), nia1nia2 (B), and QC3 (C) mutant Arabidopsis guard cells following pretreatment with opening buffer (OB). (A) Steady-state current–voltage curves for IK,in and IK,out from one guard cell of wild-type Arabidopsis recorded at intervals over 30 min after 2-h OB pretreatment. Shown are data for voltage clamp scans taken at 10 (closed circles), 20 (open circles), and 30 min (closed triangles) after impalement. 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. Current–voltage curves were fitted jointly to a Boltzmann function (solid lines) and yielding values for gmax of 3.8 and 6.3 μS cm-2, V1/2, of −181 and +1 mV, and δ of 1.9 and 1.8 for IK,in and IK,out, respectively. Insets: Current traces for time points at 30 min. Scale: 500 μA cm-2 vertical, 2 s horizontal. (B) Steady-state current–voltage curves for IK,in and IK,out from one guard cell of nia1nia2 mutant Arabidopsis recorded at intervals over 30 min after 2-h OB pretreatment. Shown are data for voltage clamp scans taken at 10 (closed circles), 20 (open circles), and 30 min (closed triangles) after impalement. Clamp voltage scans as above. Current–voltage curves were fitted jointly to a Boltzmann function (solid lines) and yielding values for gmax of 0.9 and 6.1 μS cm-2, V1/2, of −178 and +5 mV, and δ, of 1.8 and 1.8 for IK,in and IK,out, respectively. (C) Steady-state current–voltage curves for IK,in and IK,out from one guard cell of nia1nia2 mutant Arabidopsis recorded at intervals over 60 min after 2-h OB pretreatment. Shown are data for voltage clamp scans taken at 10 (closed circles), 20 (open circles), and 30 (closed triangles) 40 (open triangles), 50 (closed squares) and 60 (open squares) min after impalement. Clamp voltage scans as above. Current–voltage curves were fitted jointly to a Boltzmann function (solid lines) and yielding values for gmax of 4.1 and 4.6 μS cm-2, V1/2, -182 and −7 mV, and δ, of 1.7 and 1.9 for IK,in and IK,out, respectively. NOTE: Data analysis and curve fittings were carried out using SigmaPlot 11 (Systat Software, Inc., USA) and are reported, where appropriate, as means ± SE of n observations. Where appropriate significance was determined using Students’ T-test. Gating characteristics for IK,inand IK,outwere determined by fitting steady-state current–voltage curves to Eqn.(1)using non-linear, least-squares minimisation and the Marquardt-Levenberg algorithm[39].
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Figure 3: IK,inand IK,outof wild-type (A), nia1nia2 (B), and QC3 (C) mutant Arabidopsis guard cells following pretreatment with opening buffer (OB). (A) Steady-state current–voltage curves for IK,in and IK,out from one guard cell of wild-type Arabidopsis recorded at intervals over 30 min after 2-h OB pretreatment. Shown are data for voltage clamp scans taken at 10 (closed circles), 20 (open circles), and 30 min (closed triangles) after impalement. 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. Current–voltage curves were fitted jointly to a Boltzmann function (solid lines) and yielding values for gmax of 3.8 and 6.3 μS cm-2, V1/2, of −181 and +1 mV, and δ of 1.9 and 1.8 for IK,in and IK,out, respectively. Insets: Current traces for time points at 30 min. Scale: 500 μA cm-2 vertical, 2 s horizontal. (B) Steady-state current–voltage curves for IK,in and IK,out from one guard cell of nia1nia2 mutant Arabidopsis recorded at intervals over 30 min after 2-h OB pretreatment. Shown are data for voltage clamp scans taken at 10 (closed circles), 20 (open circles), and 30 min (closed triangles) after impalement. Clamp voltage scans as above. Current–voltage curves were fitted jointly to a Boltzmann function (solid lines) and yielding values for gmax of 0.9 and 6.1 μS cm-2, V1/2, of −178 and +5 mV, and δ, of 1.8 and 1.8 for IK,in and IK,out, respectively. (C) Steady-state current–voltage curves for IK,in and IK,out from one guard cell of nia1nia2 mutant Arabidopsis recorded at intervals over 60 min after 2-h OB pretreatment. Shown are data for voltage clamp scans taken at 10 (closed circles), 20 (open circles), and 30 (closed triangles) 40 (open triangles), 50 (closed squares) and 60 (open squares) min after impalement. Clamp voltage scans as above. Current–voltage curves were fitted jointly to a Boltzmann function (solid lines) and yielding values for gmax of 4.1 and 4.6 μS cm-2, V1/2, -182 and −7 mV, and δ, of 1.7 and 1.9 for IK,in and IK,out, respectively. NOTE: Data analysis and curve fittings were carried out using SigmaPlot 11 (Systat Software, Inc., USA) and are reported, where appropriate, as means ± SE of n observations. Where appropriate significance was determined using Students’ T-test. Gating characteristics for IK,inand IK,outwere determined by fitting steady-state current–voltage curves to Eqn.(1)using non-linear, least-squares minimisation and the Marquardt-Levenberg algorithm[39].
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.