With the exception of ALMT12, channel species other than ALMTs carry the stomatal guard cell R-type anion currents

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除了ALMT12之外，除了ALMTs以外的通道种类携带了气孔保卫细胞的R型阴离子电流。

Com exceção do ALMT12, outras espécies de canais além dos ALMTs conduzem as correntes de ânions do tipo R das células-guarda estomáticas.

Con excepción de ALMT12, las especies de canales distintas de ALMTs transportan las corrientes de aniones de tipo R de las células guardas estomáticas.

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ALMT-independent guard cell R-type anion currents

Jaślan J., Marten I., Jakobson L., Arjus T., Deeken R., Sarmiento C., De Angeli A., Brosché M., Kollist H., Hedrich R. (2023)

Justyna Jaślan, Irene Marten, Liina Jakobson, Triinu Arjus, Rosalia Deeken, Cecilia Sarmiento, Alexis De Angeli, Mikael Brosché, Hannes Kollist, Rainer Hedrich,

Department of Molecular Plant Physiology and Biophysics, Biocenter, Julius-Maximilians-Universität Würzburg (JMU), Würzburg, D-97082 Germany

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https://doi.org/10.1111/nph.19124 –

https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.19124

Summary

• Plant transpiration is controlled by stomata, with S- and R-type anion channels playing key roles in guard cell action. Arabidopsis mutants lacking the ALMT12/QUAC1 R-type anion channel function in guard cells show only a partial reduction in R-type channel currents. The molecular nature of these remaining R-type anion currents is still unclear.
• To further elucidate this, patch clamp, transcript and gas-exchange measurements were performed with wild-type (WT) and different almt mutant plants.
• The R-type current fraction in the almt12 mutant exhibited the same voltage dependence, susceptibility to ATP block and lacked a chloride permeability as the WT. Therefore, we asked whether the R-type anion currents in the ALMT12/QUAC1-free mutant are caused by additional ALMT isoforms. In WT guard cells, ALMT12, ALMT13 and ALMT14 transcripts were detected, whereas only ALMT13 was found expressed in the almt12 mutant. Substantial R-type anion currents still remained active in the almt12/13 and almt12/14 double mutants as well as the almt12/13/14 triple mutant. In good agreement, CO₂-triggered stomatal closure required the activity of ALMT12 but not ALMT13 or ALMT14.
• The results suggest that, with the exception of ALMT12, channel species other than ALMTs carry the guard cell R-type anion currents.

The nucleotide and Mg(2+) dependencies of time-dependent K(in) and K(out) channels from maize subsidiary cells were examined, showing that MgATP as well as MgADP function as channel activators

Nucleotides and Mg2+ ions differentially regulate K+ channels and non-selective cation channels present in cells forming the stomatal complex

Wolf T., Guinot D. R., Hedrich R., Dietrich P., Marten I. (2005)

Thomas Wolf, David Roger Guinot, Rainer Hedrich, Petra Dietrich, Irene Marten,

Molecular Plant Physiology and Biophysics, Julius-von-Sachs Institute for Bioscience, University of Wuerzburg, Germany.

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Plant Cell Physiol. 46(10): 1682-1689 – doi: 10.1093/pcp/pci184 – Epub 2005 Aug 4 –

https://pubmed.ncbi.nlm.nih.gov/16081526/

Abstract

Voltage-dependent inward-rectifying (K(in)) and outward-rectifying (K(out)) K(+) channels are capable of mediating K(+) fluxes across the plasma membrane. Previous studies on guard cells or heterologously expressed K(+) channels provided evidence for the requirement of ATP to maintain K(+) channel activity. Here, the nucleotide and Mg(2+) dependencies of time-dependent K(in) and K(out) channels from maize subsidiary cells were examined, showing that MgATP as well as MgADP function as channel activators. In addition to K(out) channels, these studies revealed the presence of another outward-rectifying channel type (MgC) in the plasma membrane that however gates in a nucleotide-independent manner. MgC represents a new channel type distinguished from K(out) channels by fast activation kinetics, inhibition by elevated intracellular Mg(2+) concentration, permeability for K(+) as well as for Na(+) and insensitivity towards TEA(+). Similar observations made for guard cells from Zea mays and Vicia faba suggest a conserved regulation of channel-mediated K(+) and Na(+) transport in both cell types and species.

The ABA-induced changes in time-dependent K(in) and K(out) currents from subsidiary cells are very similar to those previously described for guard cells

ABA regulation of K(+)-permeable channels in maize subsidiary cells

Wolf T., Heidelmann T., Marten I. (2006)

Thomas Wolf, Tobias Heidelmann, Irene Marten,
University of Wuerzburg, Molecular Plant Physiology and Biophysics, Julius-von-Sachs Institute for Bioscience, Julius-von-Sachs-Platz 2, D-97082 Wuerzburg, Germany.

Plant Cell Physiol. 47(10):1372-1380 – doi: 10.1093/pcp/pcl007 – Epub 2006 Sep 14 –

https://pubmed.ncbi.nlm.nih.gov/16973684/

Abstract

An antiparallel-directed potassium transport between subsidiary cells and guard cells which form the graminean stomatal complex has been proposed to drive stomatal movements in maize. To gain insights into the coordinated shuttling of K(+) ions between these cell types during stomatal closure, the effect of ABA on the time-dependent K(+) uptake and K(+) release channels as well as on the instantaneously activating non-selective cation channels (MgC) was examined in subsidiary cells. Patch-clamp studies revealed that ABA did not affect the MgC channels but differentially regulated the time-dependent K(+) channels. ABA caused a pronounced rise in time-dependent outward-rectifying K(+) currents (K(out)) at alkaline pH and decreased inward-rectifying K(+) currents (K(in)) in a Ca(2+)-dependent manner. Our results show that the ABA-induced changes in time-dependent K(in) and K(out) currents from subsidiary cells are very similar to those previously described for guard cells. Thus, the direction of K(+) transport in subsidiary cells and guard cells during ABA-induced closure does not seem to be grounded solely on the cell type-specific ABA regulation of K(+) channels.

Stomatal closure was found to be accompanied by an initial hyperpolarization and cytosolic acidification of subsidiary cells

Cell type-specific regulation of ion channels within the maize stomatal complex

Mumm P., Wolf T., Fromm J., Roelfsema M. R. G., Marten I. (2011)

Patrick Mumm, Thomas Wolf, Jörg Fromm, M Rob G Roelfsema, Irene Marten,

Plant Cell Physiol 52: 1365–1375 – doi: 10.1093/pcp/pcr082 – Epub 2011 Jun 20 –

https://pubmed.ncbi.nlm.nih.gov/21690176/

Abstract

The stomatal complex of Zea mays is composed of two pore-forming guard cells and two adjacent subsidiary cells. For stomatal movement, potassium ions and anions are thought to shuttle between these two cell types. As potential cation transport pathways, K(+)-selective channels have already been identified and characterized in subsidiary cells and guard cells. However, so far the nature and regulation of anion channels in these cell types have remained unclear. In order to bridge this gap, we performed patch-clamp experiments with subsidiary cell and guard cell protoplasts. Voltage-independent anion channels were identified in both cell types which, surprisingly, exhibited different, cell-type specific dependencies on cytosolic Ca(2+) and pH. After impaling subsidiary cells of intact maize plants with microelectrodes and loading with BCECF [(2′,7′-bis-(2-carboxyethyl)-5(and6)carboxyflurescein] as a fluorescent pH indicator, the regulation of ion channels by the cytosolic pH and the membrane voltage was further examined. Stomatal closure was found to be accompanied by an initial hyperpolarization and cytosolic acidification of subsidiary cells, while opposite responses were observed during stomatal opening. Our findings suggest that specific changes in membrane potential and cytosolic pH are likely to play a role in determining the direction and capacity of ion transport in subsidiary cells.

The plasma membrane of subsidiary cells and guard cells has to be inversely polarized in order to achieve the anti-parallel direction of K+ fluxes between these cell types during stomatal movement

Fig. 1 Isolation of subsidiary cell protoplasts from epidermal strips of Zea mays leaves. (a) Absorption of the dye neutral red indicates the viability of the stomatal complexes in isolated epidermis. The polygonal ordinary epidermal cells were not stained showing that they were destroyed upon peeling the epidermis of the leaves. (b) Magnification of a stomatal complex before enzymatic treatment. Note the chloroplast-free subsidiary cells (SC) in contrast to the chloroplast-containing guard cells (GC). (c) Subsidiary cell (SC-P) and guard cell (GC-P) protoplasts were released after enzymatic treatment of the epidermal strips for 180 min at 30°C. In line with (a) epidermal cell protoplasts were not found in the protoplast suspension. (d) Protoplasts of epidermal cells (EC-P) and mesophyll cells (MC-P) were only yielded when intact leaves were incubated in the enzyme solution. Note the 3- to 4-fold larger diameter of the epidermal cell protoplast compared to the subsidiary cell protoplast in (c).

Identification of K(+) channels in the plasma membrane of maize subsidiary cells

by Majore I., Wilhelm B., Marten I. (2002)

Ingrida Majore, Bettina Wilhelm, Irene Marten,

In Plant Cell Physiol. 43: 844–852 – https://doi.org/10.1093/pcp/pcf104 –

https://academic.oup.com/pcp/article/43/8/844/1805624

Abstract

The stomatal complex of Zea mays consists of two guard cells with the pore in between them and two flanking subsidiary cells. Both guard cells and subsidiary cells are important elements for stoma physiology because a well-coordinated transmembrane shuttle transport of potassium and chloride ions occurs between these cells during stomatal movement. To shed light upon the corresponding transport systems from subsidiary cells, subsidiary cell protoplasts were enzymatically isolated and in turn, analyzed with the patch-clamp technique. Thereby, two K⁺-selective channel types were identified in the plasma membrane of subsidiary cells. With regard to their voltage-dependent gating behavior, they may act as hyperpolarization-dependent K⁺ uptake and depolarization-activated K⁺ release channels during stomatal movement. Interestingly, the K⁺ channels from subsidiary cells and guard cells similarly responded to membrane voltage as well as to changes in the K⁺ gradient. Further, the inward- and outward-rectifying K⁺ current amplitude decreased upon a rise in the intracellular free Ca²⁺ level from 2 nM to the µM-range. The results indicate that the plasma membrane of subsidiary cells and guard cells has to be inversely polarized in order to achieve the anti-parallel direction of K⁺ fluxes between these cell types during stomatal movement.

Differential expression of K+ channels between stomatal guard cells and subsidiary cells

Differential expression of K⁺ channels between guard cells and subsidiary cells within the maize stomatal complex

Büchsenschütz K., Marten I., Becker D., Philippar K. (2), Ache P., Hedrich R. (2005)

• Kai Büchsenschütz, Irene Marten, Dirk Becker, Katrin Philippar, Peter Ache, Rainer Hedrich,

1. Molecular Plant Physiology and Biophysics, Julius-von-Sachs Institute for Bioscience, University of Wuerzburg, Wuerzburg, Germany
2. Department of Biology I, Botany III, Ludwig-Maximilians-University Muenchen, Muenchen, Germany

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In Planta 222: 968– 976 – https://doi.org/10.1007/s00425-005-0038-6 –

https://link.springer.com/article/10.1007%2Fs00425-005-0038-6

Abstract

Grass stomata are characterized by dumbbell-shaped guard cells forming a complex with a pair of specialized epidermal cells, the subsidiary cells. Stomatal movement is accomplished by a reversible exchange of potassium and chloride between these two cell types.

To gain insight into the molecular machinery involved in K⁺ transport within the stomatal complex of Zea mays, we determined the spatial and temporal expression pattern of potassium channels in the maize leaf. 

KZM2 and ZORK were isolated and identified as new members of the plant Shaker K⁺ channel family. Northern blot analysis identified fully developed leaves as the predominant site of KZM2 expression.

Following enzymatic digestion and separation of leaf tissue into epidermal, mesophyll, and vascular fractions, KZM2 and ZORK transcripts were localized in the epidermis.

Auxins can elicit stomatal opening

 

 

Plant growth hormones control voltage-dependent activity of anion channels in plasma membrane of guard cells

by Marten I., Lohse G., Hedrich R. (1991)

• Irene Marten, Gabi Lohse, Rainer Hedrich,

===

in Nature 353: 759-762 – DOI:10.1038/353758a0

https://www.nature.com/articles/353758a0

Abstract

The opening of stomatal pores in the epidermis of plant leaves is caused by an increase in turgor pressure of the guard cells as a result of the accumulation of potassium salts^1,2.

Although growth hormones have been shown to affect stomatal opening³, the transduction pathways by which growth regulators exert their effects on stomatal action are largely unknown.

Here we report that auxins can elicit stomatal opening. These phytohormones modulate anion channels^4,5 in the plasma membrane in what may be an initial step in regulated volume increase in guard cells.

Our patch-clamp experiments demonstrate that auxins can directly interact with the extracellular face of the channel. As a result, its activation potential is shifted towards the resting potential of the cell to favour transient channel opening.

Stomatal specific calcium sensitivity of high density and activity SV/TPC1 channels

 

 

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

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

Florian Rienmüller 1 , Diana Beyhl 1 , Silke Lautner 2 , Jörg Fromm 2 , Khaled A. S. Al-Rasheid 3 , Peter Ache 1 , Edward E. Farmer 4 , Irene Marten 1, Rainer Hedrich 1

1 University of Wuerzburg, Institute for Molecular Plant Physiology and Biophysics, Julius-von-Sachs Platz 2, D-97082 Wuerzburg, Germany

2 University of Hamburg, Institute for Wood Biology, Leuschnerstr. 91, 21031 Hamburg, Germany

3 King Saud University, College of Science, Zoology Department, PO Box 2455, Riyadh 11451, Saudi Arabia

4 Gene Expression Laboratory, Plant Molecular Biology, University of Lausanne, CH-1015 Lausanne, Switzerland

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in Plant Cell Physiol. 51: 1548–1554 – doi: 10.1093/pcp/pcq102 –

https://www.ncbi.nlm.nih.gov/pubmed/20630987

Abstract

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.

AtALMT12 represents an R-type anion channel required for stomatal movement

 

 

AtALMT12 represents an R-type anion channel required for stomatal movement in Arabidopsis guard cells

by Meyer S., Mumm P., Imes D., Endler A., Weder B., Al-Rasheid K. A. S., Geiger D, Marten I, Martinoia E, Hedrich R. (2010)

Stefan Meyer, Blood Donation Center Zurich, Switserland

Patrick Mumm, University of Wuerzburg, Germany

Dennis Imes, Anne Endler, Barbara Weder,

Khaled A S AL-Rasheid, King Saud University, Riyadh, Saudi Arabia

Dietmar Geiger, University of Wuerzburg, Germany

Irene Marten, University of Wuerzburg, Germany

Enrico Martinoia, University of Zurich, Switserland

Rainer Hedrich, University of Wuerzburg, Germany

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in Plant J. 63: 1054–1062. – doi: 10.1111/j.1365-313X.2010.04302.x –

https://www.ncbi.nlm.nih.gov/pubmed/20626656

Abstract

Stomatal pores formed by a pair of guard cells in the leaf epidermis control gas exchange and transpirational water loss. Stomatal closure is mediated by the release of potassium and anions from guard cells.

Anion efflux from guard cells involves slow (S-type) and rapid (R-type) anion channels. Recently the SLAC1 gene has been shown to encode the slow, voltage-independent anion channel component in guard cells. In contrast, the R-type channel still awaits identification.

Here, we show that AtALMT12, a member of the aluminum activated malate transporter family in Arabidopsis, represents a guard cell R-type anion channel. AtALMT12 is highly expressed in guard cells and is targeted to the plasma membrane.

Plants lacking AtALMT12 are impaired in dark- and CO₂ -induced stomatal closure, as well as in response to the drought-stress hormone abscisic acid. Patch-clamp studies on guard cell protoplasts isolated from atalmt12 mutants revealed reduced R-type currents compared with wild-type plants when malate is present in the bath media. Following expression of AtALMT12 in Xenopus oocytes, voltage-dependent anion currents reminiscent to R-type channels could be activated.

In line with the features of the R-type channel, the activity of heterologously expressed AtALMT12 depends on extracellular malate. Thereby this key metabolite and osmolite of guard cells shifts the threshold for voltage activation of AtALMT12 towards more hyperpolarized potentials.

R-Type channels, like voltage-dependent cation channels in nerve cells, are capable of transiently depolarizing guard cells, and thus could trigger membrane potential oscillations, action potentials and initiate long-term anion and K(+) efflux via SLAC1 and GORK, respectively.

Ion channels within the maize stomata

 

Cell type-specific regulation of ion channels within the maize stomatal complex.

by Mumm P., Wolf T., Fromm J., Roelfsema M. R. G., Marten I. (2011)

1. Patrick Mumm
2. Thomas Wolf
3. Jörg Fromm
4. M. Rob G. Roelfsema
5. Irene Marten

in Plant and Cell Physiology 52 : 1365–1375. – doi: 10.1093/pcp/pcr082 –

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

http://pcp.oxfordjournals.org/content/52/8/1365

Abstract

The stomatal complex of Zea mays is composed of two pore-forming guard cells and two adjacent subsidiary cells.

For stomatal movement, potassium ions and anions are thought to shuttle between these two cell types. As potential cation transport pathways, K⁺-selective channels have already been identified and characterized in subsidiary cells and guard cells. However, so far the nature and regulation of anion channels in these cell types have remained unclear.

In order to bridge this gap, we performed patch–clamp experiments with subsidiary cell and guard cell protoplasts. Voltage-independent anion channels were identified in both cell types which, surprisingly, exhibited different, cell-type specific dependencies on cytosolic Ca²⁺ and pH. After impaling subsidiary cells of intact maize plants with microelectrodes and loading with

BCECF [(2′,7′-bis-(2-carboxyethyl)-5(and6)carboxyflurescein] as a fluorescent pH indicator, the regulation of ion channels by the cytosolic pH and the membrane voltage was further examined.

Stomatal closure was found to be accompanied by an initial hyperpolarization and cytosolic acidification of subsidiary cells, while opposite responses were observed during stomatal opening.

Our findings suggest that specific changes in membrane potential and cytosolic pH are likely to play a role in determining the direction and capacity of ion transport in subsidiary cells.