Hormonal control of ion channel gating

Blatt M. R., Thiel G. (1993)

Annu. Rev. Plant Physiol. Mol. Biol. 44: 543–567 – https://doi.org/10.1146/annurev.pp.44.060193.002551 –

https://www.annualreviews.org/doi/10.1146/annurev.pp.44.060193.002551

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.

A critical role for ABA-mediated Ca2+ signaling as an early and overriding process leading to stomatal closure, a beneficial behavior for plants in the face of salt stress

Stomata under salt stress—what can mechanistic modeling tell us?

Thu N. B.A. , Amtmann A. , Blatt M. R., Nguyen T.-H.  (2022)

University of Glasgow – College of Medical Veterinary and Life Sciences > School of Molecular Biosciences

In: Shabala, S. (ed.) Stomata Regulation and Water Use Efficiency in Plants under Saline Soil Conditions – Series: Advances in botanical research (103) – Academic Press: Amsterdam, pp. 139-162 – ISBN 9780323912174 – doi: 10.1016/bs.abr.2022.02.012 –

https://eprints.gla.ac.uk/281348/

Abstract

Stomata are pores that form between pairs of guard cells and are commonly found in the leaf epidermis. The pores allow gaseous exchange between the inner air spaces of the leaf and the atmosphere, opening for CO2 entry to support photosynthesis and closing to reduce transpirational water loss. Guard cell membrane transport and its coordination play a central part in regulating the pore aperture. We know how guard cells respond to light, CO2 and drought with sufficient detail to model with quantitative accuracy their mechanics. By contrast, there is surprisingly little detail about the impact of salt stress on guard cells.

Here we review the topic and introduce Na+ transport within the proven OnGuard3 modeling platform to question the mechanics of stomatal responses to salinity. The analysis indicates a critical role for ABA-mediated Ca2+ signaling as an early and overriding process leading to stomatal closure, a beneficial behavior for plants in the face of salt stress.

Ca²+ and guard-cell volume in stomatal movements

Ca²+ signaling and control of guard-cell volume in stomatal movements

Blatt M. R. (2000)

Laboratory of Plant Physiology and Biophysics, Imperial College of Science, Technology and Medicine at Wye, TN25 5AH, UK.

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Curr Opin Plant Biol 3: 196–204 – PMID: 10837261 –

https://pubmed.ncbi.nlm.nih.gov/10837261/

Abstract

Stomatal guard cells are unique as a plant cell model and, because of the depth of knowledge now to hand on ion transport and its regulation, serve as an excellent model for the analysis of stimulus-response coupling in higher plants.

Parallel controls – mediated by Ca(2+), H(+) protein kinases and phosphatases – regulate the gating of the K(+) and Cl(-) channels that facilitate solute flux for stomatal movements.

A growing body of evidence now indicates that oscillations in the cytosolic free concentration of Ca(2+) contribute to a ‘signalling cassette’, which is integrated within these events through an unusual coupling with membrane voltage. Additional developments during the past two years point to events in membrane traffic that play complementary roles in stomatal control.

Research in these areas, especially, is now adding entirely new dimensions to our understanding of guard cell signalling.

The evolutionary development of grass stomata appears to have been a gradual progression

Molecular evolution of grass stomata

Chen Z.-H., Chen G., Dai F., Wang Y., Hills A., Ruan Y.-L., Zhang G., Franks P. J., Nevo E., Blatt M. R., (2017)

Trends Plant Sci 22: 124-139 – PMID:27776931 – https://doi.org/10.1016/j.tplants.2016.09.005 –

https://www.tandfonline.com/doi/full/10.1080/15592324.2017.1339858

Evolutionary trajectories of land plants have led to structurally complex and functionally active stomata for terrestrial life. A likely scenario for the emergence of active stomatal control is ‘evolutionary capture’ of key stomatal development, membrane transport, and abscisic acid signaling proteins in the divergence from liverworts to mosses.The unique morphology, development, and molecular regulation of grass stomata enable their rapid environmental response. Evolution of the molecular mechanism behind stomatal development and membrane transport has clearly drawn on conserved and sophisticated signaling networks common to stomata of all vascular plants and some mosses. Understanding this evolutionary trend will inform predictive modeling and functional manipulation of plant productivity and water use at all scales, and will benefit future efforts towards food security and ecological diversity.

Grasses began to diversify in the late Cretaceous Period and now dominate more than one third of global land area, including three-quarters of agricultural land. We hypothesize that their success is likely attributed to the evolution of highly responsive stomata capable of maximizing productivity in rapidly changing environments. Grass stomata harness the active turgor control mechanisms present in stomata of more ancient plant lineages, maximizing several morphological and developmental features to ensure rapid responses to environmental inputs. The evolutionary development of grass stomata appears to have been a gradual progression. Therefore, understanding the complex structures, developmental events, regulatory networks, and combinations of ion transporters necessary to drive rapid stomatal movement may inform future efforts towards breeding new crop varieties.

Selective block by alpha-dendrotoxin of the K+ inward rectifier at the Vicia guard cell plasma membrane

Obermeyer G., Armstrong F., Blatt M. R., (1994)

Department of Biological Sciences, University of London, Wye College, Kent, United Kingdom.

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In J. Membr. Biol. 137(3): 249-259 – doi: 10.1007/BF00232593 –

https://pubmed.ncbi.nlm.nih.gov/8182733/

Abstract

The efficacy and mechanism of alpha-dendrotoxin (DTX) block of K+ channel currents in Vicia stomatal guard cells was examined. Currents carried by inward- and outward-rectifying K+ channels were determined under voltage clamp in intact guard cells, and block was characterized as a function of DTX and external K+ (K+o) concentrations. Added to the bath, 0.1-30 nM DTX blocked the inward-rectifying K+ current (IK,in), but was ineffective in blocking current through the outward-rectifying K+ channels (IK,out) even at concentrations of 30 nM. DTX block was independent of clamp voltage and had no significant effect on the voltage-dependent kinetics for IK,in, neither altering its activation at voltages negative of -120 mV nor its deactivation at more positive voltages. No evidence was found for a use dependence to DTX action. Block of IK,in followed a simple titration function with an apparent K1/2 for block of 2.2 nM in 3 mM K+o. However, DTX block was dependent on the external K+ concentration. Raising K+o from 3 to 30 mM slowed block and resulted in a 60-70% reduction in its efficacy (apparent Ki = 10 mM in 10 nM DTX). The effect of K+ in protecting IK,in was competitive with DTX and specific for permeant cations. A joint analysis of IK,in block with DTX and K+ concentration was consistent with a single class of binding sites with a Kd for DTX of 240 pM. A Kd of 410 microM for extracellular K+ was also indicated. These results complement previous studies implicating a binding site requiring extracellular K+ (K1/2 approximately 1 mM) for IK,in activation; they parallel features of K+ channel block by DTX and related peptide toxins in many animal cells, demonstrating the sensitivity of plant plasma membrane K+ channels to nanomolar toxin concentrations under physiological conditions; the data also highlight one main difference: in the guard cells, DTX action appears specific to the K+ inward rectifier.

Signaling elements in stomatal guard cell ion channel control

Co-ordination of signaling elements in guard cell ion channel control

by Grabov A., Blatt M. R. (1998)

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In J. Exp. Bot. 49: 351–360 – 

https://www.jstor.org/stable/23695968?seq=1#page_scan_tab_contents

Abstract

Fine regulation of solutes transport across the guard cell plasma membrane for osmotic modulation is essential for the maintenance of the proper stomatal aperture in response to environmental stimuli.

The major osmotica, K+, Cl- and malate are transported through selective ion channels in the plasma membrane and tonoplast of guard cells.

To date, a number of ion channels have been shown to operate in the guard cell plasma membrane: outwardly- and inwardly-rectifying K+ channels (IK,in and IK,out), slowly- and rapid-activating anion channels, and stretch-activated non-selective channels. Slow and fast vacuolar channels (SV and FV) and voltage-independent K+-selective (VK) channels have been found at the guard cell tonoplast. On the molecular level, the regulation of the stomatal aperture is achieved by precise spatial and temporal co-ordination of channel activities through a network of signalling cascades that can be triggered, among others, by plant hormones. ABA and auxin as regulators of stomatal aperture have received most attention to date.

It is clear now that the effect of these hormones on ion channels is mediated by second messengers pHi and [Ca2+]i. The effect of ABA is generally associated with increases in pHi, while auxin acidifies the cytosol. Both of these hormones may induce elevation in [Ca2+]i.

Phosphorylation is another important factor in cellular signalling. The ABI1 gene encoding a 2C-type protein phosphatase has been shown to be a key element of ABA-dependent cascades.

Plasma membrane voltage is also an important component of signalling and channel control, and has recently been shown to influence cytosolic-free [Ca2+]. Thus, transduction of these, and associated cytoplasmic signals is clearly non-linear, and is probably important for providing a plasticity of cellular response to external and environmental stimuli. Understanding the interdependence and hierarchy of signalling elements now presents a major challenge for research in plant biology.

Voltage control and membrane transport in stomatal guard cells

Membrane transport in stomatal guard cells: the importance of voltage control

by Thiel G., MacRobbie E. A., Blatt M. R. (1992)

Botany School, University of Cambridge, Cambridge, England

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In Journal of Membrance Biology 126: 1-18 – https://doi.org/10.1007/BF00233456 –

https://link.springer.com/article/10.1007/BF00233456

http://www.esalq.usp.br/lepse/imgs/conteudo_thumb/Membrane-Transport-in-Stomata-in-Guard-Cells-The-Importance-of-Voltage-Control.pdf

Abstract

Potassium uptake and export in the resting conditions and in response to the phytohormone abscisic acid (ABA) were examined under voltage clamp in guard cells of Vicia faba L. In 0.1 mm external K⁺ (with 5 mm Ca²⁺-HEPES, pH 7.4) two distinct transport states could be identified based on the distribution of the free-running membrane voltage(V _M ) data in conjunction with the respective I-V and G-V relations. One state was dominated by passive diffusion (mean V _M = −143± 4 mV), the other (mean V _M = −237± 10 mV) exhibited an appreciable background of primary H⁺ transport activity. In the presence of pump activity the free-running membrane voltage was negative of the respective K⁺equilibrium potential (E_K⁺), in 3 and 10 mm external K⁺. In these cases V _M was also negative of the activation voltage for the inward rectifying K⁺ current, thus creating a strong bias for passive K⁺ uptake through inward-rectifying K⁺ channels. In contrast, when pump activity was absent V _M was situated positive of E_K⁺ and cells revealed a bias for K⁺ efflux. Occasionally spontaneous voltage transitions were observed during which cells switched between the two states. Rapid depolarizations were induced in cells with significant pump activity upon adding 10 μm ABA to the medium. These depolarizations activated current through outward-rectifying K⁺ channels which was further amplified in ABA by a rise in the ensemble channel conductance. Current-voltage characteristics recorded before and during ABA treatments revealed concerted modulations in current passage through at least four distinct transport processes, results directly comparable to one previous study (Blatt, M.R., 1990, Planta 180:445) carried out with guard cells lacking detectable primary pump activity. Comparative analyses of guard cells in each case are consistent with depolarizations resulting from the activation of an inward-going, as yet unidentified current, rather than an ABA-induced fall in H⁺-ATPase output. Also observed in a number of cells was an inward-directed current which activated in ABA over a narrow range of voltages positive of -150 mV; this and additional features of the current suggest that it may reflect the ABA-dependent activation of an anion channel previously characterized in Vicia guard cell protoplasts, but rule out its function as the primary mechanism for initial depolarization. The analyses also yield indirect evidence for a rise in cytoplasmic Ca²⁺ activity in ABA, as well as for a K⁺ current distinct from the dominant inward and outward-rectifying K⁺ channels, but neither support nor discount a role for Ca²⁺influx in depolarizing the membrane. A striking similarity was found for the modulation of inward currents either in response to ABA or after spontaneous depolarizations. This renders the possibility of an agonist (i.e., ABA) activated Ca²⁺ current across the plasma membrane as trigger for the voltage transitions unlikely.

The potential of enhancing stomatal kinetics to improve water use efficiency without penalty in carbon fixation

Optogenetic manipulation of stomatal kinetics improves carbon assimilation, water use, and growth

by Papanatsiou M., Petersen J., Henderson L., Wang Y., Christie J. M., Blatt M. R. (2019)

1. M. Papanatsiou¹,², 
2. J. Petersen²,*, 
3. L. Henderson², 
4. Y. Wang¹,³, 
5. J. M. Christie²,†,‡, 
6. M. R. Blatt¹,²,³,†,‡

1. Laboratory of Plant Physiology and Biophysics, Institute of Molecular, Cell and Systems Biology, University of Glasgow, University Avenue, Glasgow G12 8QQ, UK.
2. Plant Science Group, Institute of Molecular, Cell and Systems Biology, University of Glasgow, University Avenue, Glasgow G12 8QQ, UK.
3. Institute of Crop Science, College of Agriculture and Biotechnology, Zijingang Campus, Zhejiang University, Hangzhou 310058, China.

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In Science 363, Issue 6434, 1456-1459 – DOI: 10.1126/science.aaw0046 –

http://science.sciencemag.org/content/363/6434/1456

Speeding up stomatal responses

A plant’s cellular metabolism rapidly adjusts to changes in light conditions, but its stomata—pores that allow gas exchange in leaves—are slower to respond. Because of the lagging response, photosynthesis is less efficient, and excess water is lost through the open pores. Papanatsiou et al. introduced a blue light–responsive ion channel into stomata of the small mustard plant Arabidopsis. The channel increased the rate of stomata opening and closing in response to light. The engineered plants produced more biomass, especially in the fluctuating light conditions typical of outdoor growth.

Abstract

Stomata serve dual and often conflicting roles, facilitating carbon dioxide influx into the plant leaf for photosynthesis and restricting water efflux via transpiration. Strategies for reducing transpiration without incurring a cost for photosynthesis must circumvent this inherent coupling of carbon dioxide and water vapor diffusion.

We expressed the synthetic, light-gated K⁺ channel BLINK1 in guard cells surrounding stomatal pores in Arabidopsis to enhance the solute fluxes that drive stomatal aperture.

BLINK1 introduced a K⁺conductance and accelerated both stomatal opening under light exposure and closing after irradiation. Integrated over the growth period, BLINK1 drove a 2.2-fold increase in biomass in fluctuating light without cost in water use by the plant.

Thus, we demonstrate the potential of enhancing stomatal kinetics to improve water use efficiency without penalty in carbon fixation.

PYR/PYL/RCAR receptor coupling to the activation by ABA of plasma membrane Ca(2+) channels through ROS, affecting [Ca(2+)]i and its regulation of stomatal closure

PYR/PYL/RCAR abscisic acid receptors regulate K⁺ and Cl⁻ channels through reactive oxygen species‐mediated activation of Ca²⁺ channels at the plasma membrane of intact Arabidopsis guard cells

by Wang Y., Chen Z. H., Zhang B., Hills A., Blatt M. R. ( 2013)

Laboratory of Plant Physiology and Biophysics, University of Glasgow, Glasgow G12 8QQ, United Kingdom.

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In Plant Physiol 163: 566– 577 – DOI: 10.1104/pp.113.219758 –

https://www.ncbi.nlm.nih.gov/pubmed/23899646?dopt=Abstract

Abstract

The discovery of the START family of abscisic acid (ABA) receptors places these proteins at the front of a protein kinase/phosphatase signal cascade that promotes stomatal closure.

The connection of these receptors to Ca(2+) signals evoked by ABA has proven more difficult to resolve, although it has been implicated by studies of the pyrbactin-insensitive pyr1/pyl1/pyl2/pyl4 quadruple mutant. One difficulty is that flux through plasma membrane Ca(2+) channels and Ca(2+) release from endomembrane stores coordinately elevate cytosolic free Ca(2+) concentration ([Ca(2+)]i) in guard cells, and both processes are facilitated by ABA.

Here, we describe a method for recording Ca(2+) channels at the plasma membrane of intact guard cells of Arabidopsis (Arabidopsis thaliana).

We have used this method to resolve the loss of ABA-evoked Ca(2+) channel activity at the plasma membrane in the pyr1/pyl1/pyl2/pyl4 mutant and show the consequent suppression of [Ca(2+)]i increases in vivo. The basal activity of Ca(2+) channels was not affected in the mutant; raising the concentration of Ca(2+) outside was sufficient to promote Ca(2+) entry, to inactivate current carried by inward-rectifying K(+) channels and to activate current carried by the anion channels, both of which are sensitive to [Ca(2+)]i elevations. However, the ABA-dependent increase in reactive oxygen species (ROS) was impaired. Adding the ROS hydrogen peroxide was sufficient to activate the Ca(2+) channels and trigger stomatal closure in the mutant.

These results offer direct evidence of PYR/PYL/RCAR receptor coupling to the activation by ABA of plasma membrane Ca(2+) channels through ROS, thus affecting [Ca(2+)]i and its regulation of stomatal closure.