An aberrant K+ channel behavior in stomatal guard cells

Sensitivity to abscisic acid of guard-cell K+ channels is suppressed by abi1-1, a mutant Arabidopsis gene encoding a putative protein phosphatase

Armstrong F., Leung J., Grabov A., Brearley J., Giraudat J., Blatt M. R., (1995)

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Proc Natl Acad Sci U S A. 92(21): 9520-9524 – doi: 10.1073/pnas.92.21.9520 – PMID: 7568166 – PMCID: PMC40833 –

https://pubmed.ncbi.nlm.nih.gov/7568166/

Abstract

Abscisic acid (ABA) modulates the activities of three major classes of ion channels–inward- and outward-rectifying K+ channels (IK,in and IK,out, respectively) and anion channels–at the guard-cell plasma membrane to achieve a net efflux of osmotica and stomatal closure. Disruption of ABA sensitivity in wilty abi1-1 mutants of Arabidopsis and evidence that this gene encodes a protein phosphatase suggest that protein (de)-phosphorylation contributes to guard-cell transport control by ABA. To pinpoint the role of ABI1, the abi1-1 dominant mutant allele was stably transformed into Nicotiana benthamiana and its influence on IK,in, IK,out, and the anion channels was monitored in guard cells under voltage clamp. Compared with guard cells from wild-type and vector-transformed control plants, expression of the abi1-1 gene was associated with 2- to 6-fold reductions in IK,out and an insensitivity of both IK,in and IK,out to 20 microM ABA. In contrast, no differences between control and abi1-1 transgenic plants were observed in the anion current or its response to ABA. Parallel measurements of intracellular pH (pHi) using the fluorescent dye 2′,7′-bis(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) in every case showed a 0.15- to 0.2-pH-unit alkalinization in ABA, demonstrating that the transgene was without effect on the pHi signal that mediates in ABA-evoked K+ channel control. In guard cells from the abi1-1 transformants, normal sensitivity of both K+ channels to and stomatal closure in ABA was recovered in the presence of 100 microM H7 and 0.5 microM staurosporine, both broad-range protein kinase antagonists. These results demonstrate an aberrant K+ channel behavior–including channel insensitivity to ABA-dependent alkalinization of pHi–as a major consequence of abi1-1 action and implicate AB11 as part of a phosphatase/kinase pathway that modulates the sensitivity of guard-cell K+ channels to ABA-evoked signal cascades.

ABA and anion channel kinetics in stomata

 

Alteration of anion channel kinetics in wild-type and abi1-1 transgenic Nicotiana benthamiana guard cells by abscisic acid.

by Grabov A., Leung J., Giraudat J.,  Blatt M. R.(1997)

in Plant J.12, 203–213. –

Wiley Online Library | PubMed | CAS | CrossRefMedlineWeb of Science 

http://www.ncbi.nlm.nih.gov/pubmed/9263461

Abstract

The influence of the plant water-stress hormone abscisic acid (ABA) on anion channel activity and its interaction with protein kinase and phosphatase antagonists was examined in stomatal guard cells of wild-type Nicotiana benthamiana L. and of transgenic plants expressing the dominant-negative (mutant) Arabidopsis abi1-1 protein phosphatase.

Intact guard cells were impaled with double-barrelled micro-electrodes and membrane current was recorded under voltage clamp in the presence of 15 mM CsCl and 15 mM tetraethylammonium chloride (TEA-Cl) to eliminate K+ channel currents. Under these conditions, the free-running voltage was situated close to 0 mV (+9 +/- 6 mV, n = 18) and the membrane under voltage clamp was dominated by anion channel current (ICl) as indicated from tall current reversal near the expected chloride equilibrium potential, current sensitivity to the anion channel blockers 9-anthracene carboxylic acid and niflumic acid, and by its voltage-dependent kinetics.

Pronounced activation of ICl was recorded on stepping from a conditioning voltage of -250 mV to voltages between -30 and +50 mV, and the current deactivated with a voltage-dependent halftime at more negative voltages (tau approximately equal to 0.3 sec at -150 mV). Challenge with 20 microM ABA increased the steady-state current conductance, gCl, near 0 mV by 1.2- to 2.6-fold and at -150 mV by 4.5- to sixfold with a time constant of 40 +/- 4 sec, and it slowed ICl deactivation as much as fourfold at voltages near -50 mV, introducing two additional voltage-sensitive kinetic components to these current relaxations.

Neither the steady-state and kinetic characteristics of ICl nor its sensitivity to ABA were influenced by H7 or staurosporine, both broad-range protein kinase antagonists. However, the protein phosphatase 1/2A antagonist calyculin A mimicked the effects of ABA on gCl and current relaxations on its own and exhibited a synergistic interaction with ABA, enhancing ICl sensitivity to ABA three- to four-fold.

Quantitatively similar current characteristics were recorded from guard cells of abi1-1 transgenic N. benthamiana, indicating that the abi1-1 protein phosphatase does not influence the anion current or its response to ABA directly.

These results demonstrate that ABA stimulates ICl and modulates its voltage sensitivity. Furthermore, they show that ABA promotes ICl, either by introducing additional long-lived states of the channel or by activating a second anion channel with similar permeation characteristics but with a very long dwell time in the open state.

Overall, the data are broadly consistent with the view that ABA action engenders coordinate control of ICl together with guard cell K+ channels to effect solute loss and stomatal closure.

OST1 kinase activity and function in stomata

 

 

Identification of features regulating OST1 kinase activity and OST1 function in guard cells.

by Belin C., de Franco P. O., Bourbousse C., Chaignepain S., Schmitter J. M., Vavasseur A., Giraudat J., Barbier-Brygoo H., Thomine S. (2006)

in Plant Physiol 141:1316–1327. – doi: http://dx.doi.org/10.1104/pp.106.079327

Abstract/FREE Full Text

http://www.plantphysiol.org/content/141/4/1316.abstract?ijkey=d1eea710947fd1da948ca953573f7de31cf27137&keytype2=tf_ipsecsha

Abstract

The phytohormone abscisic acid (ABA) mediates drought responses in plants and, in particular, triggers stomatal closure.

Snf1-related kinase 2 (SnRK2) proteins from several plant species have been implicated in ABA-signaling pathways. In Arabidopsis (Arabidopsis thaliana) guard cells, OPEN STOMATA 1 (OST1)/SRK2E/SnRK2-6 is a critical positive regulator of ABA signal transduction.

A better understanding of the mechanisms responsible for SnRK2 protein kinase activation is thus a major goal toward understanding ABA signal transduction.

Here, we report successful purification of OST1 produced in Escherichia coli: The protein is active and autophosphorylates. Using mass spectrometry, we identified five target residues of autophosphorylation in recombinant OST1.

Sequence analysis delineates two conserved boxes located in the carboxy-terminal moiety of OST1 after the catalytic domain: the SnRK2-specific box (glutamine-303 to proline-318) and the ABA-specific box (leucine-333 to methionine-362). Site-directed mutagenesis and serial deletions reveal that serine (Ser)-175 in the activation loop and the SnRK2-specific box are critical for the activity of recombinant OST1 kinase.

Targeted expression of variants of OST1 kinase in guard cells uncovered additional features that are critical for OST1 function in ABA signaling, although not required for OST1 kinase activity: Ser-7, Ser-18, and Ser-29 and the ABA-specific box. Ser-7, Ser-18, Ser-29, and Ser-43 represent putative targets for regulatory phosphorylation and the ABA-specific box may be a target for the binding of signaling partners in guard cells.

OST1 protein kinase and stomatal movement

 

 

Arabidopsis OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production.

by Mustilli A. C., Merlot S., Vavasseur A., Fenzi F., Giraudat J. (2002)

in Plant Cell 14:3089–3099. –

Abstract/FREE Full Text – CrossRef, PubMed, CAS, Web of Science® – ISI, CAS, PubMed, Article

http://www.plantcell.org/lens/plantcell/14/12/3089

Abstract

During drought, the plant hormone abscisic acid (ABA) triggers stomatal closure, thus reducing water loss. Using infrared thermography, we isolated two allelic Arabidopsis mutants (ost1-1 and ost1-2) impaired in the ability to limit their transpiration upon drought. These recessive ost1 mutations disrupted ABA induction of stomatal closure as well as ABA inhibition of light-induced stomatal opening.

By contrast, the ost1 mutations did not affect stomatal regulation by light or CO2, suggesting that OST1 is involved specifically in ABA signaling. The OST1 gene was isolated by positional cloning and was found to be expressed in stomatal guard cells and vascular tissue.

In-gel assays indicated that OST1 is an ABA-activated protein kinase related to the Vicia faba ABA-activated protein kinase (AAPK). Reactive oxygen species (ROS) were shown recently to be an essential intermediate in guard cell ABA signaling. ABA-induced ROS production was disrupted in ost1 guard cells, whereas applied H2O2 or calcium elicited the same degree of stomatal closure in ost1 as in the wild type.

These results suggest that OST1 acts in the interval between ABA perception and ROS production. The relative positions of ost1 and the other ABA-insensitive mutations in the ABA signaling network (abi1-1, abi2-1, and gca2) are discussed.

OST1 kinase activity and OST1 function in guard cells

 

 

Identification of features regulating OST1 kinase activity and OST1 function in guard cells.

by Belin C., de Franco P. O., Bourbousse C., Chaignepain S., Schmitter J. M., Vavasseur A., Giraudat J., Barbier-Brygoo H., Thomine S. (2006)

in Plant Physiol. 2006 Aug;141(4):1316-27. Epub 2006 Jun 9.

http://www.ncbi.nlm.nih.gov/pubmed/16766677

Abstract

The phytohormone abscisic acid (ABA) mediates drought responses in plants and, in particular, triggers stomatal closure. Snf1-related kinase 2 (SnRK2) proteins from several plant species have been implicated in ABA-signaling pathways. In Arabidopsis (Arabidopsis thaliana) guard cells, OPEN STOMATA 1 (OST1)/SRK2E/SnRK2-6 is a critical positive regulator of ABA signal transduction.

A better understanding of the mechanisms responsible for SnRK2 protein kinase activation is thus a major goal toward understanding ABA signal transduction.

Here, we report successful purification of OST1 produced in Escherichia coli: The protein is active and autophosphorylates. Using mass spectrometry, we identified five target residues of autophosphorylation in recombinant OST1. Sequence analysis delineates two conserved boxes located in the carboxy-terminal moiety of OST1 after the catalytic domain: the SnRK2-specific box (glutamine-303 to proline-318) and the ABA-specific box (leucine-333 to methionine-362). Site-directed mutagenesis and serial deletions reveal that serine (Ser)-175 in the activation loop and the SnRK2-specific box are critical for the activity of recombinant OST1 kinase.

Targeted expression of variants of OST1 kinase in guard cells uncovered additional features that are critical for OST1 function in ABA signaling, although not required for OST1 kinase activity: Ser-7, Ser-18, and Ser-29 and the ABA-specific box. Ser-7, Ser-18, Ser-29, and Ser-43 represent putative targets for regulatory phosphorylation and the ABA-specific box may be a target for the binding of signaling partners in guard cells.

An aberrant K+ channel behavior in stomata

 

 

Sensitivity to abscisic acid of guard cell K⁺ channels is suppressed by abi1-1, a mutant Arabidopsis gene encoding a putative protein phosphatase.

by Armstrong F.,Leung J., Grabov A., Brearley J., Giraudat J., Blatt M.R.(1995)

Blatt M.R.

in Proc. Natl Acad. Sci. USA, 92, 9520–9524. – DOI: 10.1073/pnas.92.21.9520

CrossRef | PubMed | CAS | ADS

http://www.pnas.org/content/92/21/9520

http://www.ncbi.nlm.nih.gov/pubmed/7568166

http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=wiley&version=1%2E0&coi=1%3aCAS%3a528%3aDyaK2MXoslCnurk%253D&md5=5ba6995c093f8524104e33099f953f76

http://adsabs.harvard.edu/abs/1995PNAS…92.9520A

Abstract

Abscisic acid (ABA) modulates the activities of three major classes of ion channels–inward- and outward-rectifying K+ channels (IK,in and IK,out, respectively) and anion channels–at the guard-cell plasma membrane to achieve a net efflux of osmotica and stomatal closure.

Disruption of ABA sensitivity in wilty abi1-1 mutants of Arabidopsis and evidence that this gene encodes a protein phosphatase suggest that protein (de)-phosphorylation contributes to guard-cell transport control by ABA.

To pinpoint the role of ABI1, the abi1-1 dominant mutant allele was stably transformed into Nicotiana benthamiana and its influence on IK,in, IK,out, and the anion channels was monitored in guard cells under voltage clamp. Compared with guard cells from wild-type and vector-transformed control plants, expression of the abi1-1 gene was associated with 2- to 6-fold reductions in IK,out and an insensitivity of both IK,in and IK,out to 20 microM ABA.

In contrast, no differences between control and abi1-1 transgenic plants were observed in the anion current or its response to ABA. Parallel measurements of intracellular pH (pHi) using the fluorescent dye 2′,7′-bis(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) in every case showed a 0.15- to 0.2-pH-unit alkalinization in ABA, demonstrating that the transgene was without effect on the pHi signal that mediates in ABA-evoked K+ channel control.

In guard cells from the abi1-1 transformants, normal sensitivity of both K+ channels to and stomatal closure in ABA was recovered in the presence of 100 microM H7 and 0.5 microM staurosporine, both broad-range protein kinase antagonists.

These results demonstrate an aberrant K+ channel behavior–including channel insensitivity to ABA-dependent alkalinization of pHi–as a major consequence of abi1-1 action and implicate AB11 as part of a phosphatase/kinase pathway that modulates the sensitivity of guard-cell K+ channels to ABA-evoked signal cascades.

The role of abi1-1, a mutant Arabidopsis gene, in stomata

 

Sensitivity to abscisic acid of guard-cell K+ channels is suppressed by abi1-1, a mutant Arabidopsis gene encoding a putative protein phosphatase

by Armstrong F., Leung J., Grabov A., Brearley J., Giraudat J., Blatt M.R.  (1995)

in Proceedings of the National Academy of Sciences, USA 1995;92:9520-9524. –

Abstract/FREE Full Text

Abstract

Abscisic acid (ABA) modulates the activities of three major classes of ion channels–inward- and outward-rectifying K+ channels (IK,in and IK,out, respectively) and anion channels–at the guard-cell plasma membrane to achieve a net efflux of osmotica and stomatal closure. Disruption of ABA sensitivity in wilty abi1-1 mutants of Arabidopsis and evidence that this gene encodes a protein phosphatase suggest that protein (de)-phosphorylation contributes to guard-cell transport control by ABA.

To pinpoint the role of ABI1, the abi1-1 dominant mutant allele was stably transformed into Nicotiana benthamiana and its influence on IK,in, IK,out, and the anion channels was monitored in guard cells under voltage clamp. Compared with guard cells from wild-type and vector-transformed control plants, expression of the abi1-1 gene was associated with 2- to 6-fold reductions in IK,out and an insensitivity of both IK,in and IK,out to 20 microM ABA.

In contrast, no differences between control and abi1-1 transgenic plants were observed in the anion current or its response to ABA. Parallel measurements of intracellular pH (pHi) using the fluorescent dye 2′,7′-bis(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) in every case showed a 0.15- to 0.2-pH-unit alkalinization in ABA, demonstrating that the transgene was without effect on the pHi signal that mediates in ABA-evoked K+ channel control. In guard cells from the abi1-1 transformants, normal sensitivity of both K+ channels to and stomatal closure in ABA was recovered in the presence of 100 microM H7 and 0.5 microM staurosporine, both broad-range protein kinase antagonists.

These results demonstrate an aberrant K+ channel behavior–including channel insensitivity to ABA-dependent alkalinization of pHi–as a major consequence of abi1-1 action and implicate AB11 as part of a phosphatase/kinase pathway that modulates the sensitivity of guard-cell K+ channels to ABA-evoked signal cascades.

See the text: PNAS

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