ABA depolarizes guard cells in intact plants



ABA depolarizes guard cells in intact plants, through a transient activation of R- and S-type anion channels.

by Roelfsema M. R. G., Levchenko V., Hedrich R. (2004)

Julius-von-Sachs-Institut für Biowissenschaften, Lehrstuhl für Molekulare Pflanzenphysiologie und Biophysik, Universität Würzburg, Julius-von-Sachs-Platz 2, D-97082, Germany.


in Plant Journal 37: 578588. – doi:10.1111/j.1365-313X.2003.01985.x pmid:14756768 

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During drought, the plant hormone abscisic acid (ABA) induces rapid stomatal closure and in turn reduces transpiration. Stomatal closure is accompanied by large ion fluxes across the plasma membrane, carried by K+ and anion channels.

We recorded changes in the activity of these channels induced by ABA, for guard cells of intact Vicia faba plants. Guard cells in their natural environment were impaled with double-barrelled electrodes, and ABA was applied via the leaf surface. In 45 out of 85 cells tested, ABA triggered a transient depolarization of the plasma membrane. In these cells, the membrane potential partially recovered in the presence of ABA; however, a full recovery of the membrane potentials was only observed after removal of ABA.

Repetitive ABA responses could be evoked in single cells, but the magnitude of the response varied from one hormone application to the other. The transient depolarization correlated with the activation of anion channels, which peaked 5 min after introduction of the stimulus.

In guard cells with a moderate increase in plasma membrane conductance (DeltaG < 5 nS), ABA predominantly activated voltage-independent (slow (S)-type) anion channels. During strong responses (DeltaG > 5 nS), however, ABA activated voltage-dependent (rapid (R)-type) in addition to S-type anion channels.

We conclude that the combined activation of these two channel types leads to the transient depolarization of guard cells. The nature of this ABA response correlates with the transient extrusion of Cl- from guard cells and a rapid but confined reduction in stomatal aperture.



Modulation of stomatal movement by apoplastic factors.



Studying guard cells in the intact plant: modulation of stomatal movement by apoplastic factors.

by Roelfsema M. R. G., Hedrich R. (2002)

in New Phytol. 153, 425–431. – doi: 10.1046/j.1469-8137.2002.00344.x –

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DOI: 10.1046/j.0028-646X.2001.Documedoc.doc.x – 



Here, we discuss why guard cells in intact plants respond to environmental signals in a different way than guard cells in epidermal strips, or protoplasts thereof.

In intact leaves stomatal opening is counteracted by epidermal cells that press against the guard cells. Changes in the turgor of epidermal cells therefore can alter the stomatal aperture.

In addition, stomatal opening may be modulated by the solute composition of the guard cell wall. Changes in apoplastic K+, Cl and Ca2+ occur after light–dark transitions, but not in such a way that it would support stomatal opening.

Organic anions may play a role, since they enhance the open probability of anion channels in the plasma membrane.

Furthermore, studies with auxin-resistant and abscisic acid-insensitive mutants show that light-induced stomatal opening is modulated by these hormones.

Using the newly developed method in which guard cells in the intact plant are impaled with double-barreled electrodes, the role of these apoplastic factors now can be studied on single guard cells that are still in their natural environment.

K+ channel contributes significantly to the uptake and release of K+ by guard cells during stomatal movement



Potassium-selective single channels in guard cell protoplasts of Vicia faba.

Schroeder J. I., Hedrich R., Fernandez J. M. (1984)


*Department of Membrane Biophysics, Max–Planck-Institut für biophysikalische Chemie, D-3400 Göttingen, FRG
Pflanzenphysiologisches Institut, Universität Göttingen, D-3400 Göttingen, FRG

in Nature, 312, 361362 – doi:10.1038/312361a0  –

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In plants, stomata control the gaseous exchange between the intercellular spaces of the leaf and the atmosphere. Fluxes of ions, in particular potassium fluxes, across the membranes of the guard cells produce changes in turgor of the guard cells which in turn result in the opening and closing of the stomatal pore1,2.

The molecular mechanisms involved in the uptake or release of ions in guard cells are poorly understood2. Cell-free membrane patches of the plasmalemma, isolated following patch-clamp techniques3–5, allow, for the first time in higher plant cells, the separation of the electrical properties of the plasmalemma from those of the tonoplast.

We have applied these techniques to study the properties of single-ion channels in the plasmalemma of guard cell protoplasts of Vicia faba (broad bean).

Predominantly a cation-selective channel was observed, which showed a high selectivity for K+, with a permeability ratio P K+/P Na+ of 11:1 and a single-channel conductance of 37 pS(=37 × 10;−12 −1) in symmetrical 225 mM KC1 solutions.

We estimate that this K+ channel contributes significantly to the uptake and release of K+ by guard cells during stomatal movement.


Guard cell-specific calcium sensitivity


Guard cell-specific calcium sensitivity of high density and activity SV/TPC1 channels. 

by Rienmuller F., Beyhl D., Lautner S., Fromm J., Al-Rasheid K. A. S., Ache P., Farmer E. E., Marten I., Hedrich R. (2010)

University of Wuerzburg, Institute for Molecular Plant Physiology and Biophysics, Wuerzburg, Germany.

in Plant and Cell Physiology 5115481554 – doi: 10.1093/pcp/pcq102. Epub 2010 Jul 14. – 



The slow vacuolar (SV) channel, a Ca2+-regulated vacuolar cation conductance channel, in Arabidopsis thaliana is encoded by the single-copy gene AtTPC1.

Although loss-of-function tpc1 mutants were reported to exhibit a stoma phenotype, knowledge about the underlying guard cell-specific features of SV/TPC1 channels is still lacking.

Here we demonstrate that TPC1 transcripts and SV current density in guard cells were much more pronounced than in mesophyll cells. Furthermore, the SV channel in motor cells exhibited a higher cytosolic Ca2+ sensitivity than in mesophyll cells.

These distinct features of the guard cell SV channel therefore probably account for the published stomatal phenotype of tpc1-2.

A voltagedependent anion channel in the plasma membrane of guard cells (stomata)


Identification and modulation of a voltagedependent anion channel in the plasma membrane of guard cells by high-affinity ligands.

by Marten I., Zeilinger C., Redhead C., Landry D. W., Al-Awqati Q., Hedrich R. (1992)

in EMBO J 11: 3569-3575 –



Guard cell anion channels (GCAC1) catalyze the release of anions across the plasma membrane during regulated volume decrease and also seem to be involved in the targeting of the plant growth hormones auxins.

We have analyzed the modulation and inhibition of these voltage-dependent anion channels by different anion channel blockers. Ethacrynic acid, a structural correlate of an auxin, caused a shift in activation potential and simultaneously a transient increase in the peak current amplitude, whereas other blockers shifted and blocked the voltage-dependent activity of the channel.

Comparison of dose-response curves for shift and block imposed by the inhibitor, indicate two different sites within the channel which interact with the ligand. The capability to inhibit GCAC1 increases in a dose-dependent manner in the sequence: probenecid less than A-9-C less than ethacrynic acid less than niflumic acid less than IAA-94 less than NPPB.

All inhibitors reversibly blocked the anion channel from the extracellular side. Channel block on the level of single anion channels is characterized by a reduction of long open transitions into flickering bursts, indicating an interaction with the open mouth of the channel. IAA-23, a structural analog of IAA-94, was used to enrich ligand-binding polypeptides from the plasma membrane of guard cells by IAA-23 affinity chromatography. From this protein fraction a 60 kDa polypeptide crossreacted specifically with polyclonal antibodies raised against anion channels isolated from kidney membranes.

In contrast to guard cells, mesophyll plasma membranes were deficient in voltage-dependent anion channels and lacked crossreactivity with the antibody.

Stilbene derivatives and the plasma membrane anion channel of stomata


Modulation and block of the plasma membrane anion channel of guard cells by stilbene derivatives.

by Marten I.Busch H.Raschke K.Hedrich R. (1993)

Rainer_Hedrich.png – University of Würzburg



in Eur Biophys J 21:403408. –

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An anion channel in the plasma membrane of guard cells (GCAC1) provides a regulatory element for the voltage-dependent release of anions during stomatal closure (Keller et al. 1989) as well as excitability (Hedrich et al. 1990). Recognition sites for plant growth hormones on the extracellular surface of GCAC1 further indicate that this channel may also serve as a transduction element in hormone signaling (Marten et al. 1991 a).

Stilbene derivatives were used to study the inhibitor-structure channel-function relationship of GCAC1. We have analyzed the activity, voltage-gate and kinetics of this channel as affected by stilbenes. The stilbene derivatives SITS and DNDS caused a shift in activation potential and a decrease in the peak current amplitude.

Channel block through the action of DIDS, on the other hand, was not accompanied by a shift in voltage-dependence. Differences in the dose-dependence of the two effects give clues to the presence of channel sites responsible for gate-shifting and block. The ability to inhibit anion currents (K(d)) increased in the sequence: SITS (4 muM) < DNDS (0.5 muM) < DIDS (0.2 muM). All inhibitors reversibly blocked the anion channel from the extracellular side. Channel block on the level of single anion-channels is characterized by a reduction of long open-transitions into flickering bursts and a decrease in channel amplitude.

The anion channel in stomata and auxin


Anions modify the response of guard cell anion channels to auxin.

by Lohse G., Hedrich R. (1995)

Lohse G.

Hedrich Rainer, Universität Würzburg csm_0129hedrich1-w_00e0ff5f8c

in Planta 197: 546-552, 1995. – doi:10.1007/BF00196677 –



The anion channel in the guard-cell plasma membrane of Vicia faba, GCAC1, possesses recognition sites for the plant growth hormone auxin at the extracellular mouth of the channel (Marten et al. 1991, Nature 353:759-762).

Using the patch-clamp technique we could demonstrate that auxins induced a shift of the voltage dependence of the anion channel to hyperpolarized potentials; the shift was attenuated during an increase in the extracellular chloride concentration, indicating that chloride shields the hormone-binding site. The auxin-induced shift was concentration-dependent, characterized by a Michaelis-Menten type of behaviour with a half saturation constant (Km) of about 10 μM naphthalene-1-acetic acid (1-NAA) in the presence of 2 mM Cl and 12 μM in 80 mM Cl.

In the presence of malate, another gating modulator of GCAC1, auxins were less effective, indicating that both ligands compete for common sites. Inactive auxins with respect to stomatal opening or stimulation of the plasma membrane H+-ATPase, such as 2-NAA, modulated the activation threshold and kinetics of GCAC1 similar to the active form 1-NAA. At a concentration of 100 μM 2-NAA the peak-current potential shifted by about 30 mV more negative.