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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

Effect of pre-treatment with opening buffer (OB) on I anionin wild-type Arabidopsis . Steady-state current–voltage curves for Ianion from one guard cell recorded after 2-h pretreatment with OB. Current–voltage curves for Ianion are not corrected for background. Rundown in this cell was evident only after 65 min. Data shown are taken from voltage clamp scans at 10 (closed circles), 20 (open circles), and 30 (inverted closed triangles) and 40 min (open triangles) after the impalement. Conditioning voltage was +50 mV with 10-s steps to voltages between +50 mV and −220 mV. Inset: Raw current traces for recordings at 10, 20 and 30 min cross-referenced by symbol. Scale: vertical, 300 μA cm-2; horizontal, 5 s.
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Figure 5: Effect of pre-treatment with opening buffer (OB) on I anionin wild-type Arabidopsis . Steady-state current–voltage curves for Ianion from one guard cell recorded after 2-h pretreatment with OB. Current–voltage curves for Ianion are not corrected for background. Rundown in this cell was evident only after 65 min. Data shown are taken from voltage clamp scans at 10 (closed circles), 20 (open circles), and 30 (inverted closed triangles) and 40 min (open triangles) after the impalement. Conditioning voltage was +50 mV with 10-s steps to voltages between +50 mV and −220 mV. Inset: Raw current traces for recordings at 10, 20 and 30 min cross-referenced by symbol. Scale: vertical, 300 μA cm-2; horizontal, 5 s.

Mentions: To date, only Forestier et al. [28] reported Ianion in intact Arabidopsis stomatal guard cells, although components of Ianion have been identified with the SLAC1 and ALMT12 gene products [41-44]. We recorded Ianion in over 95% of cases from 158 guard cells in RB2 with current similar to past measurements from Arabidopsis, Vicia and tobacco [28,35,40]. The mean membrane voltage of −9.9 ± 1.6 mV in RB2 was also comparable to those recorded in these previous studies. We found no appreciable difference between guard cells with or without OB pretreatment (Additonal file 1: Table S 1) but, again, pretreatment prolonged the timeframe for Ianion recordings and experiments frequently extended over periods of one hour (Table 2 and Figure 5). Thus OB pretreatment improved the stability of Ianion recordings much as it did for those of IK,in and IK,out.


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

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

Effect of pre-treatment with opening buffer (OB) on I anionin wild-type Arabidopsis . Steady-state current–voltage curves for Ianion from one guard cell recorded after 2-h pretreatment with OB. Current–voltage curves for Ianion are not corrected for background. Rundown in this cell was evident only after 65 min. Data shown are taken from voltage clamp scans at 10 (closed circles), 20 (open circles), and 30 (inverted closed triangles) and 40 min (open triangles) after the impalement. Conditioning voltage was +50 mV with 10-s steps to voltages between +50 mV and −220 mV. Inset: Raw current traces for recordings at 10, 20 and 30 min cross-referenced by symbol. Scale: vertical, 300 μA cm-2; horizontal, 5 s.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Effect of pre-treatment with opening buffer (OB) on I anionin wild-type Arabidopsis . Steady-state current–voltage curves for Ianion from one guard cell recorded after 2-h pretreatment with OB. Current–voltage curves for Ianion are not corrected for background. Rundown in this cell was evident only after 65 min. Data shown are taken from voltage clamp scans at 10 (closed circles), 20 (open circles), and 30 (inverted closed triangles) and 40 min (open triangles) after the impalement. Conditioning voltage was +50 mV with 10-s steps to voltages between +50 mV and −220 mV. Inset: Raw current traces for recordings at 10, 20 and 30 min cross-referenced by symbol. Scale: vertical, 300 μA cm-2; horizontal, 5 s.
Mentions: To date, only Forestier et al. [28] reported Ianion in intact Arabidopsis stomatal guard cells, although components of Ianion have been identified with the SLAC1 and ALMT12 gene products [41-44]. We recorded Ianion in over 95% of cases from 158 guard cells in RB2 with current similar to past measurements from Arabidopsis, Vicia and tobacco [28,35,40]. The mean membrane voltage of −9.9 ± 1.6 mV in RB2 was also comparable to those recorded in these previous studies. We found no appreciable difference between guard cells with or without OB pretreatment (Additonal file 1: Table S 1) but, again, pretreatment prolonged the timeframe for Ianion recordings and experiments frequently extended over periods of one hour (Table 2 and Figure 5). Thus OB pretreatment improved the stability of Ianion recordings much as it did for those of IK,in and IK,out.

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