Pflügers Arch – Eur J Physiol (1998) 436:920–927
© Springer-Verlag 1998
O R I G I N A L A RT I C L E
Cyrille Forestier · François Bouteau Nathalie Leonhardt · Alain Vavasseur
Pharmacological properties of slow anion currents in intact guard cells of Arabidopsis. Application of the discontinuous single-electrode voltage-clamp to different species Received: 5 January 1998 / Received after revision: 26 April 1998 / Accepted: 2 July 1998
Abstract More electrophysiological studies have been carried out on guard cells than on any other cell type of vascular plants. The characterization of their ion channels has been achieved using mainly the whole-cell patch-clamp technique applied to guard-cell protoplasts. The aim of this study was to obtain recordings of ion channel currents in intact guard cells and especially of slow anion channels of Arabidopsis thaliana, a species of fundamental genetic interest. Application of the discontinuous single-electrode voltage-clamp technique enabled the first characterization of K+ currents in Commelina communis and of slow anion currents in C. communis and A. thaliana in intact guard cells to be made. Inward K+ channels from A. thaliana were inhibited by external application of tetraethylammonium (TEA) or Ca2+. In the presence of K+ channel blockers, slow anion channel currents were elicited in almost all guard cells tested and were confirmed by the application of anion channel blockers. In A. thaliana, only anthracene-9 carboxylic acid was able to inhibit slow anion currents, to promote stomatal opening in the dark and to reverse the effect of 25 µM abscisic acid under light. Use of a single microelectrode and preservation of cell integrity make this technique well suited for the study of ion channel regulation in species that have guard cell protoplasts with which it is difficult to form good seals. Key words Anion channel inhibitors · Arabidopsis thaliana · Commelina communis · Intact guard cell · Slow anion and potassium currents · Vicia faba C. Forestier (✉) · N. Leonhardt · A. Vavasseur CEA Cadarache, DSV–DEVM, Laboratoire de Bioénergétique Cellulaire, BP1, F-13108, St Paul-lez-Durance, Cedex France e-mail:
[email protected] Tel.: +33-4-42253048, Fax: +33-4-42254656 F. Bouteau Laboratoire de Physiologie Cellulaire et Moléculaire du Stress Végétal, Université Denis Diderot, Paris 7, case 70–69, 2 place Jussieu, F-75251 Paris Cedex 05, France
Introduction Guard cells are located at the end of the transpiration stream within the plant and their control of stomatal aperture is crucial for minimizing water loss from the leaf tissues while balancing the requirement of CO2 exchange for photosynthesis. As far back as 1968, Fisher [18] discovered that the osmotically driven movement of guard cells is correlated with substantial transport of K+ ions through the plasma membrane. Since then, application of the patch-clamp technique to guard cell protoplasts [37] or use of conventional microelectrodes [3, 4] has provided information at the molecular level on ion fluxes, allowing the characterization of numerous ion channels in terms of their regulation by nucleotides, Ca2+, pH, membrane potential as well as membrane tension (for review see [2]). The benefits of using the whole-cell patch-clamp technique when studying guard cell protoplasts are its high resolution of current measurements, resulting from the tight seal with small whole cells, and the chemical access to both sides of the membrane. This advantage is exemplified by the primary identification of all ion channels in guard cells using this technique. Unfortunately, obtention of protoplasts suitable for patch-clamp experiments depends on species. Three different species are mainly used to study signal transduction in stomatal guard cells [44]. 1. Vicia faba has been used most for guard cell electrophysiological studies. Characterization and function of K+ [42] and anion [33] channels in the regulation of stomatal function has been reported in patch-clamp studies. 2. Commelina communis is a species widely used for biochemical approaches due to the ease with which guard cell protoplasts of high purity can be obtained. Moreover, pharmacological experiments are generally conducted with C. communis since stomatal apertures in epidermal peels are easy to measure. However, no current characterization in this species is available, despite some interesting physiological characteristics such as its high sensitivity towards Ca2+ [40]
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3. Arabidopsis thaliana has become the “model plant” for genetics. The identification of numerous mutants reinforces the interest in this species in physiological studies. Only recently, the first description of the involvement of slow anion channels in the stomatal response to abscisic acid (ABA) in A. thaliana wildtype and abi mutants was given in a report of a patchclamp study [29]. Another approach to analysis of ion channels in intact cells is the microelectrode voltage-clamp method. This technique allows reliable long-term recordings of the free-running membrane potential with very low levels of perfusion of the cell interior [9]; however, it does not permit the study of single-channel activity. Until now, all current recordings made from intact guard cells have been conducted using a two-electrode voltage-clamp system (TEVC [5]). Subtle differences in the results obtained using patch-clamp and TEVC can be found, e.g. the modulation of the outward K+ channel with respect to external K+ concentration [7, 34]. However, the results obtained using both techniques compare very favourably. TEVC has allowed original studies, such as the effects of CO2 on K+ and anion channels in V. faba [11], or investigations of the impact of the abi1 mutation on guard cell K+ channel currents [1]. Although TEVC has been successfully used on V. faba guard cells for years, no-one has yet published data concerning anion currents in A. thaliana or in C. communis intact guard cells. Discontinuous single-electrode voltage-clamp (dSEVC) is a technique originally developed for small neurons for which penetration by two electrodes is too traumatic. Only a few reports on its use in plant cells exist [10] despite the fact that, theoretically, it is well suited to small and symplastically isolated cells such as Arabidopsis guard cells. In this study, we report on the first successful application of dSEVC to intact guard cells from V. faba, C. communis and A. thaliana. K+ and anion currents were recorded and we present the first pharmacological characterization of slow anion channel activity in A. thaliana guard cells published to date.
periments using A. thaliana were conducted as previously described [25]. All values in this report are given with the standard error of the mean (95% confidence interval). Electrophysiological measurements Microelectrodes were pulled on a Narishige PE-6 vertical puller with borosilicate capillary glass (Clark GC-150F, Phymep, Paris, France) and coated with beeswax before use in order to reduce electrode capacitance. The resistance of the microelectrodes, filled with 300 mM K+ acetate, 1 mM KCl, pH 7.5 to minimize salt leakages, was 83±6 MΩ (n=85). Bath solution or ion-channel modulators were perfused by gravity into a 300-µl recording chamber containing the epidermis. The bath solution was exchanged (volume 10×) in less than 1 min. A pellet (E205, Phymep, Paris, France) was used as a reference electrode in the recording chamber. Voltage-clamp measurements were carried out using dSEVC [17] to record the whole-cell currents from intact guard cells. In this technique, both current-passing and voltage-recording are achieved using the same microelectrode. Interactions between the two tasks are prevented by multiplexing techniques which ensure that the voltage recorded by the microelectrode tip is sampled and saved only after the current-induced voltage drop across the microelectrode has decayed to a negligible value. In this study, the commutation rate was 2–5 kHz. This value lies between the time constant of the electrode (equivalent frequency >10 kHz) after optimization of the pipette parameters and the membrane time constant (equivalent frequency 10 s) was clearly different from that described above for outward K+ currents (
