Dynamic regulation of guard cell anion channels by cytosolic free Ca2+ concentration and protein phosphorylation

In guard cells, activation of anion channels (I_anion) is an early event leading to stomatal closure. Activation of I_anion has been associated with abscisic acid (ABA) and its elevation of the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). However, the dynamics of the action of [Ca²⁺]_i on I_anion has never been established, despite its importance for understanding the mechanics of stomatal adaptation to stress. We have quantified the [Ca²⁺]_i dynamics of I_anion in Vicia faba guard cells, measuring channel current under a voltage clamp while manipulating and recording [Ca²⁺]_i using Fura‐2 fluorescence imaging. We found that I_anion rises with [Ca²⁺]_i only at concentrations substantially above the mean resting value of 125 ± 13 nm , yielding an apparent K_d of 720 ± 65 nm and a Hill coefficient consistent with the binding of three to four Ca²⁺ ions to activate the channels. Approximately 30% of guard cells exhibited a baseline of I_anion activity, but without a dependence of the current on [Ca²⁺]_i. The protein phosphatase antagonist okadaic acid increased this current baseline over twofold. Additionally, okadaic acid altered the [Ca²⁺]_i sensitivity of I_anion, displacing the apparent K_d for [Ca²⁺]_i to 573 ± 38 nm . These findings support previous evidence for different modes of regulation for I_anion, only one of which depends on [Ca²⁺]_i, and they underscore an independence of [Ca²⁺]_i from protein (de‐)phosphorylation in controlling I_anion. Most importantly, our results demonstrate a significant displacement of I_anion sensitivity to higher [Ca²⁺]_i compared with that of the guard cell K⁺ channels, implying a capacity for variable dynamics between net osmotic solute uptake and loss.     

Cyclic adenosine 5′‐diphosphoribose (cADPR) cyclic guanosine 3′,5′‐monophosphate positively function in Ca2+ elevation in methyl jasmonate‐induced stomatal closure,cADPR is required for methyl jasmonate‐induced ROS accumulation NO production in guard cells

Methyl jasmonate (MeJA) signalling shares several signal components with abscisic acid (ABA) signalling in guard cells. Cyclic adenosine 5′‐diphosphoribose (cADPR) and cyclic guanosine 3′,5′‐monophosphate (cGMP) are second messengers in ABA‐induced stomatal closure. In order to clarify involvement of cADPR and cGMP in MeJA‐induced stomatal closure in Arabidopsis thaliana (Col‐0), we investigated effects of an inhibitor of cADPR synthesis, nicotinamide (NA), and an inhibitor of cGMP synthesis, LY83583 (LY, 6‐anilino‐5,8‐quinolinedione), on MeJA‐induced stomatal closure. Treatment with NA and LY inhibited MeJA‐induced stomatal closure. NA inhibited MeJA‐induced reactive oxygen species (ROS) accumulation and nitric oxide (NO) production in guard cells. NA and LY suppressed transient elevations elicited by MeJA in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) in guard cells. These results suggest that cADPR and cGMP positively function in [Ca²⁺]_cyt elevation in MeJA‐induced stomatal closure, are signalling components shared with ABA‐induced stomatal closure in Arabidopsis, and that cADPR is required for MeJA‐induced ROS accumulation and NO production in Arabidopsis guard cells.     

Increases in cytosolic Ca2+ are not required for abscisic acid-inhibition of inward K+ currents in guard cells of Vicia faba L.

 The inward K⁺ channels (I_Kin) of guard cells are inhibited upon application of abscisic acid (ABA). It has been postulated that I_Kin inhibition requires an elevation in cytosolic free Ca²⁺ levels ([Ca²⁺]_c) because: (i) experimental increases in [Ca²⁺] _c can mimic the ABA effect, and; (ii) ABA can trigger an elevation of [Ca²⁺]_c in guard cells. However, not all guard cells respond to ABA with a [Ca²⁺]_c increase, and the magnitude of the increases that do occur is variable. Therefore, an obligate role for Ca²⁺ in the regulation of downstream effectors of ABA response, such as the I_Kin channels, remains in question. In this study, we developed a methodology for simultaneous patch clamping and confocal ratiometric Ca²⁺ imaging of Vicia faba L. guard-cell protoplasts. This allowed us to directly assess the relationship between ABA-induced changes in [Ca²⁺]_c and I_Kin inhibition. In the presence of extracellular Ca²⁺, the extent of [Ca²⁺]_c elevation correlated with the extent of I_Kin inhibition. However, upon chelation of either extracellular Ca²⁺, [Ca²⁺]_c, or both, extracellular Ca²⁺ and [Ca²⁺]_c, [Ca²⁺]_c elevation did not occur in response to ABA yet I_Kin currents were still strongly inhibited. These data illustrate that Ca²⁺-independent regulation is involved in ABA-inhibition of stomatal opening processes. Received: 17 September 1999 / Accepted: 26 October 1999     

Involvement of extracellular oxidative burst in salicylic acid-induced stomatal closure in Arabidopsis

Salicylic acid (SA), a ubiquitous phenolic phytohormone, is involved in many plant physiological processes including stomatal movement. We analysed SA‐induced stomatal closure, production of reactive oxygen species (ROS) and nitric oxide (NO), cytosolic calcium ion ([Ca²⁺]_cyt) oscillations and inward‐rectifying potassium (K⁺_in) channel activity in Arabidopsis. SA‐induced stomatal closure was inhibited by pre‐treatment with catalase (CAT) and superoxide dismutase (SOD), suggesting the involvement of extracellular ROS. A peroxidase inhibitor, SHAM (salicylhydroxamic acid) completely abolished SA‐induced stomatal closure whereas neither an inhibitor of NADPH oxidase (DPI) nor atrbohD atrbohF mutation impairs SA‐induced stomatal closures. 3,3′‐Diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) stainings demonstrated that SA induced H₂O₂ and O₂^– production. Guard cell ROS accumulation was significantly increased by SA, but that ROS was suppressed by exogenous CAT, SOD and SHAM. NO scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO) suppressed the SA‐induced stomatal closure but did not suppress guard cell ROS accumulation whereas SHAM suppressed SA‐induced NO production. SA failed to induce [Ca²⁺]_cyt oscillations in guard cells whereas K⁺_in channel activity was suppressed by SA. These results indicate that SA induces stomatal closure accompanied with extracellular ROS production mediated by SHAM‐sensitive peroxidase, intracellular ROS accumulation and K⁺_in channel inactivation.     

The Arabidopsis heterotrimeric G‐protein β subunit,AGB1, is required for guard cell calcium sensing and calcium‐induced calcium release

Cytosolic calcium concentration ([Ca²⁺]_cyt) and heterotrimeric G‐proteins are universal eukaryotic signaling elements. In plant guard cells, extracellular calcium (Ca_o) is as strong a stimulus for stomatal closure as the phytohormone abscisic acid (ABA), but underlying mechanisms remain elusive. Here, we report that the sole Arabidopsis heterotrimeric Gβ subunit, AGB1, is required for four guard cell Ca_o responses: induction of stomatal closure; inhibition of stomatal opening; [Ca²⁺]_cyt oscillation; and inositol 1,4,5‐trisphosphate (InsP3) production. Stomata in wild‐type Arabidopsis (Col) and in mutants of the canonical Gα subunit, GPA1, showed inhibition of stomatal opening and promotion of stomatal closure by Ca_o. By contrast, stomatal movements of agb1 mutants and agb1/gpa1 double‐mutants, as well as those of the agg1agg2 Gγ double‐mutant, were insensitive to Ca_o. These behaviors contrast with ABA‐regulated stomatal movements, which involve GPA1 and AGB1/AGG3 dimers, illustrating differential partitioning of G‐protein subunits among stimuli with similar ultimate impacts, which may facilitate stimulus‐specific encoding. AGB1 knockouts retained reactive oxygen species and NO production, but lost YC3.6‐detected [Ca²⁺]_cyt oscillations in response to Ca_o, initiating only a single [Ca²⁺]_cyt spike. Experimentally imposed [Ca²⁺]_cyt oscillations restored stomatal closure in agb1. Yeast two‐hybrid and bimolecular complementation fluorescence experiments revealed that AGB1 interacts with phospholipase Cs (PLCs), and Ca_o induced InsP3 production in Col but not in agb1. In sum, G‐protein signaling via AGB1/AGG1/AGG2 is essential for Ca_o‐regulation of stomatal apertures, and stomatal movements in response to Ca_o apparently require Ca²⁺‐induced Ca²⁺ release that is likely dependent on Gβγ interaction with PLCs leading to InsP3 production.     

K252a-sensitive protein kinases but not okadaic acid-sensitive protein phosphatases regulate methyl jasmonate-induced cytosolic Ca2+ oscillation in guard cells of Arabidopsis thaliana

Methyl jasmonate (MeJA) induces stomatal closure similar to abscisic acid (ABA), and MeJA signaling in guard cells shares some signal components with ABA signaling. As part of this process, MeJA as well as ABA induce the elevation and oscillation of cytosolic free-calcium concentrations ([Ca²⁺]_cyt) in guard cells. While abscisic acid-induced [Ca²⁺]_cyt oscillation has been extensively studied, MeJA-induced [Ca²⁺]_cyt oscillation is less well understood. In this study, we investigated the effects of K252a (a broad-range protein kinase inhibitor) and okadaic acid (OA, a protein phosphatase 1 and 2A inhibitor) on MeJA-induced [Ca²⁺]_cyt oscillation in guard cells of Arabidopsis thaliana ecotype Columbia expressing the Ca²⁺ reporter yellow cameleon 3.6. The protein kinase inhibitor K252a abolished MeJA-induced stomatal closure and reduced MeJA-elicited [Ca²⁺]_cyt oscillation. The protein phosphatase inhibitor OA, on the other hand, did not inhibit these processes. These results suggest that MeJA signaling involves activation of K252a-sensitive protein kinases upstream of [Ca²⁺]_cyt oscillation but not activation of an OA-sensitive protein phosphatase in guard cells of A. thaliana ecotype Columbia.     

Negative regulation of abscisic acid-induced stomatal closure by glutathione in Arabidopsis

We found that glutathione (GSH) is involved in abscisic acid (ABA)-induced stomatal closure. Regulation of ABA signaling by GSH in guard cells was investigated using an Arabidopsis mutant, cad2-1, that is deficient in the first GSH biosynthesis enzyme, γ-glutamylcysteine synthetase, and a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene (CDNB). Glutathione contents in guard cells decreased along with ABA-induced stomatal closure. Decreasing GSH by both the cad2-1 mutation and CDNB treatment enhanced ABA-induced stomatal closure. Glutathione monoethyl ester (GSHmee) restored the GSH level in cad2-1 guard cells and complemented the stomatal phenotype of the mutant. Depletion of GSH did not significantly increase ABA-induced production of reactive oxygen species in guard cells and GSH did not affect either activation of plasma membrane Ca²⁺-permeable channel currents by ABA or oscillation of the cytosolic free Ca²⁺ concentration induced by ABA. These results indicate that GSH negatively modulates a signal component other than ROS production and Ca²⁺ oscillation in ABA signal pathway of Arabidopsis guard cells.     

Osmotic stress and abscisic acid reduce cytosolic calcium activities in roots of Arabidopsis thaliana*

Cytosolic Ca²⁺· ([Ca²⁺]_i, and elongation growth were measured in the roots of Arabidopsis thaliana. Exposure of plant tissues to high NaCl and abscisic acid (ABA) concentrations results in a reduction in the rate of growth, but the mechanism by which growth is inhibited is not understood. Both NaCl and ABA treatments are known to influence [Ca²⁺]_i, and in this study we measured the effects of salinity and ABA on [Ca²⁺]_i in cells from the meristematic region of Arabidopsis roots. The Ca²⁺-sensitive dye Fura-2 and ratiometric techniques were used to measure [Ca²⁺]_i in cells of the root meristem region. Resting [Ca²⁺]_i was found to be between 100 and 200 μmol m^?3 in roots of untreated plants. Resting [Ca²⁺]_i changed in response to changes in the [Ca²⁺] surrounding growing roots. An increase of external [Ca²⁺] increased [Ca²⁺]_i; conversely, a decrease of external [Ca²⁺] decreased [Ca²⁺]_i. Exposure of roots to NaCl caused a rapid reduction of [Ca²⁺]_i, a response that was proportional to the external NaCl concentration. Thus, as the NaCl concentration was increased, [Ca²⁺]_i in root meristematic cells decreased. Root elongation was also inhibited in proportion to the external NaCl concentration, with maximal inhibition occurring at 120 mol m^?3 NaCl. The [Ca²⁺]_i of root meristem cells also changed in response to ABA, and the magnitude of the effect of ABA was dependent upon ABA concentration. Treatment with 0.2 mmol m^?3 ABA caused a momentary increase in [Ca²⁺]_i followed by a decrease after 15 min, but 10 mmol m^?3 ABA caused an immediate decline in [Ca²⁺]_i. There was a strong positive correlation between [Ca²⁺]_i and root elongation rates. Experiments with the ABA-deficient Arabidopsis mutant aba-3 indicated that the reduction in [Ca²⁺]_i brought about by NaCl was unlikely to be mediated via changes in endogenous ABA. Experiments with solutes such as sorbitol, KCl and NaNO₃ indicated that the effects of NaCl could be mimicked by other solutes and was not specific for NaCl.     

Two guard cell mitogen‐activated protein kinases,MPK9 and MPK12, function in methyl jasmonate‐induced stomatal closure in Arabidopsis thaliana

Methyl jasmonate (MeJA) and abscisic acid (ABA) signalling cascades share several signalling components in guard cells. We previously showed that two guard cell‐preferential mitogen‐activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signalling in Arabidopsis thaliana. In this study, we examined whether these two MAP kinases function in MeJA signalling using genetic mutants for MPK9 and MPK12 combined with a pharmacological approach. MeJA induced stomatal closure in mpk9‐1 and mpk12‐1 single mutants as well as wild‐type plants, but not in mpk9‐1 mpk12‐1 double mutants. Consistently, the MAPKK inhibitor PD98059 inhibited the MeJA‐induced stomatal closure in wild‐type plants. MeJA elicited reactive oxygen species (ROS) production and cytosolic alkalisation in guard cells of the mpk9‐1, mpk12‐1 and mpk9‐1 mpk12‐1 mutants, as well in wild‐type plants. Furthermore, MeJA triggered elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in the mpk9‐1 mpk12‐1 double mutant as well as wild‐type plants. Activation of S‐type anion channels by MeJA was impaired in mpk9‐1 mpk12‐1. Together, these results indicate that MPK9 and MPK12 function upstream of S‐type anion channel activation and downstream of ROS production, cytosolic alkalisation and [Ca²⁺]_cyt elevation in guard cell MeJA signalling, suggesting that MPK9 and MPK12 are key regulators mediating both ABA and MeJA signalling in guard cells.     

Early ABA Signaling Events in Guard Cells

The plant hormone abscisic acid (ABA) regulates a wide variety of plant physiological and developmental processes, particularly responses to environmental stress, such as drought. In response to water deficiency, plants redistribute foliar ABA and/or upregulate ABA synthesis in roots, leading to roughly a 30-fold increase in ABA concentration in the apoplast of stomatal guard cells. The elevated ABA triggers a chain of events in guard cells, causing stomatal closure and thus preventing water loss. Although the molecular nature of ABA receptor(s) remains unknown, considerable progress in the identification and characterization of its downstream signaling elements has been made by using combined physiological, biochemical, biophysical, molecular, and genetic approaches. The measurable events associated with ABA-induced stomatal closure in guard cells include, sequentially, the production of reactive oxygen species (ROS), increases in cytosolic free Ca²⁺ levels ([Ca²⁺]_i), activation of anion channels, membrane potential depolarization, cytosolic alkalinization, inhibition of K⁺ influx channels, and promotion of K⁺ efflux channels. This review provides an overview of the cellular and molecular mechanisms underlying these ABA-evoked signaling events, with particular emphasis on how ABA triggers an “electronic circuitry” involving these ionic components.     

Two guard cell‐preferential MAPKs,MPK9 and MPK12, regulate YEL signalling in Arabidopsis guard cells

We report that two mitogen‐activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate abscisic acid (ABA)‐induced stomatal closure in Arabidopsis thaliana. Yeast elicitor (YEL) induced stomatal closure accompanied by intracellular reactive oxygen species (ROS) accumulation and cytosolic free calcium concentration ([Ca²⁺]_cyt) oscillation. In this study, we examined whether these two MAP kinases are involved in YEL‐induced stomatal closure using MAPKK inhibitors, PD98059 and U0126, and MAPK mutants, mpk9, mpk12 and mpk9 mpk12. Both PD98059 and U0126 inhibited YEL‐induced stomatal closure. YEL induced stomatal closure in the mpk9 and mpk12 mutants but not in the mpk9 mpk12 mutant, suggesting that a MAPK cascade involving MPK9 and MPK12 functions in guard cell YEL signalling. However, YEL induced extracellular ROS production, intracellular ROS accumulation and cytosolic alkalisation in the mpk9, mpk12 and mpk9 mpk12 mutants. YEL induced [Ca²⁺]_cyt oscillations in both wild type and mpk9 mpk12 mutant. These results suggest that MPK9 and MPK12 function redundantly downstream of extracellular ROS production, intracellular ROS accumulation, cytosolic alkalisation and [Ca²⁺]_cyt oscillation in YEL‐induced stomatal closure in Arabidopsis guard cells and are shared with ABA signalling.     

The trafficking protein SYP121 of Arabidopsis connects programmed stomatal closure and K⁺ channel activity with vegetative growth

The vesicle‐trafficking protein SYP121 (SYR1/PEN1) was originally identified in association with ion channel control at the plasma membrane of stomatal guard cells, although stomata of the Arabidopsis syp121 loss‐of‐function mutant close normally in ABA and high Ca²⁺. We have now uncovered a set of stomatal phenotypes in the syp121 mutant that reduce CO₂ assimilation, slow vegetative growth and increase water use efficiency in the whole plant, conditional upon high light intensities and low relative humidity. Stomatal opening and the rise in stomatal transpiration of the mutant was delayed in the light and following Ca²⁺‐evoked closure, consistent with a constitutive form of so‐called programmed stomatal closure. Delayed reopening was observed in the syp121, but not in the syp122 mutant lacking the homologous gene product; the delay was rescued by complementation with wild‐type SYP121 and was phenocopied in wild‐type plants in the presence of the vesicle‐trafficking inhibitor Brefeldin A. K⁺ channel current that normally mediates K⁺ uptake for stomatal opening was suppressed in the syp121 mutant and, following closure, its recovery was slowed compared to guard cells of wild‐type plants. Evoked stomatal closure was accompanied by internalisation of GFP‐tagged KAT1 K⁺ channels in both wild‐type and syp121 mutant guard cells, but their subsequently recycling was slowed in the mutant. Our findings indicate that SYP121 facilitates stomatal reopening and they suggest that K⁺ channel traffic and recycling to the plasma membrane underpins the stress memory phenomenon of programmed closure in stomata. Additionally, they underline the significance of vesicle traffic for whole‐plant water use and biomass production, tying SYP121 function to guard cell membrane transport and stomatal control.     

Role of calcium in the modulation of Vicia guard cell potassium channels by abscisic acid: A patch-clamp study

There is evidence for a role of increased cytoplasmic Ca²⁺ in the stomatal closure induced by abscisic acid (ABA), but two points of controversy remain the subject of vigorous debate—the universality of Ca²⁺ as a component of the signaling chain, and the source of the increased Ca²⁺, whether influx across the plasmalemma, or release from internal stores. We have addressed these questions by patch-clamp studies on guard cell protoplasts of Vicia faba, assessing the effects of ABA in the presence and absence of external Ca²⁺, and of internal Ca²⁺ buffers to control levels of cytoplasmic Ca²⁺. We show that ABA-induced reduction of the K⁺ inward rectifier can occur in the absence of external Ca²⁺, but is abolished when Ca²⁺ buffers are present inside the cell. Thus, some minimum level of cytoplasmic Ca²⁺ is a necessary component of the signaling chain by which ABA decreases the K⁺ inward rectifier in stomatal guard cells, thus preventing stomatal opening. Release of Ca²⁺ from internal stores is capable of mediating the response, in the absence of any Ca²⁺ influx from the extracellular medium. The work also shows that enhancement of the K⁺ outward rectifier by ABA is Ca²⁺ independent, and that other signaling mechanisms must be involved. A role for internal pH, as suggested by H.R. Irving, C.A. Gehring and R.W. Parish (Proc. Natl. Acad. Sci. USA 89:1790–1794, 1990) and M.R. Blatt (J. Gen. Physiol. 99:615–644, 1992), is an attractive working hypothesis.     

The Roles of CATALASE2 in Abscisic Acid Signaling in Arabidopsis Guard Cells

We investigated the roles of catalase (CAT) in abscisic acid (ABA)-induced stomatal closure using a cat2 mutant and an inhibitor of CAT, 3-aminotriazole (AT). Constitutive reactive oxygen species (ROS) accumulation due to the CAT2 mutation and AT treatment did not affect stomatal aperture in the absence of ABA, whereas ABA-induced stomatal closure, ROS production, and [Ca²⁺]_cyt oscillation were enhanced.     

Calcium ions as second messengers in guard cell signal transduction

Ca²⁺ is a ubiquitous second messenger in plant cell signalling. In this review we consider the role of Ca²⁺-based signal transduction in stomatal guard cells focusing on three important areas: (1) the regulation of guard cell turgor relations and the control of gene expression in guard cells, (2) the control of specificity in Ca²⁺ signalling, (3) emerging technologies and new approaches for studying intracellular signalling. Stomatal apertures alter in response to a wide array of environmental stimuli as a result of changes in guard cell turgor. For example, the plant hormone abscisic acid (ABA) stimulates a reduction in stomatal aperture through a decrease in guard cell turgor. Furthermore, guard cells have been shown to be competent to relay an ABA signal from its site of perception to the nucleus. An increase in the concentration of cytosolic free Ca²⁺ ([Ca²⁺]₁) is central to the mechanisms underlying ABA-induced changes in guard cell turgor. We describe a possible model of Ca²⁺-based ABA signal transduction during stomatal closure and discuss recent evidence which suggests that Ca²⁺ is also involved in ABA nuclear signal transduction. Many other environmental stimuli which affect stomatal apertures, in addition to ABA, induce an increase in guard cell [Ca²⁺]₁) This raises questions regarding how increases in [Ca²⁺]₁) can be a common component in the signal transduction pathways by which stimuli cause both stomatal opening and closure. We discuss several mechanisms of increasing the amount of information contained within the Ca²⁺ signal, including encoding information in a stimulus-specific Ca²⁺ signal or Ca²⁺ signature', the concept of the ‘physiological address’ of the cell, and the use of other second messengers. We conclude by addressing the emerging technologies and new approaches which can be used in conjunction with guard cells to dissect further the molecular mechanisms of Ca²⁺-mediated signalling in plants.     

De-regulated expression of the plant glutamate receptor homolog AtGLR3.1 impairs long-term Ca2+-programmed stomatal closure

Cytosolic Ca²⁺ ([Ca²⁺]_cyt) mediates diverse cellular responses in both animal and plant cells in response to various stimuli. Calcium oscillation amplitude and frequency control gene expression. In stomatal guard cells, [Ca²⁺]_cyt has been shown to regulate stomatal movements, and a defined window of Ca²⁺ oscillation kinetic parameters encodes necessary information for long‐term stomatal movements. However, it remains unknown how the encrypted information in the cytosolic Ca²⁺ signature is decoded to maintain stomatal closure. Here we report that the Arabidopsis glutamate receptor homolog AtGLR3.1 is preferentially expressed in guard cells compared to mesophyll cells. Furthermore, over‐expression of AtGLR3.1 using a viral promoter resulted in impaired external Ca²⁺‐induced stomatal closure. Cytosolic Ca²⁺ activation of S‐type anion channels, which play a central role in Ca²⁺‐reactive stomatal closure, was normal in the AtGLR3.1 over‐expressing plants. Interestingly, AtGLR3.1 over‐expression did not affect Ca²⁺‐induced Ca²⁺ oscillation kinetics, but resulted in a failure to maintain long‐term ‘Ca²⁺‐programmed’ stomatal closure when Ca²⁺ oscillations containing information for maintaining stomatal closure were imposed. By contrast, prompt short‐term Ca²⁺‐reactive closure was not affected in AtGLR3.1 over‐expressing plants. In wild‐type plants, the translational inhibitor cyclohexamide partially inhibited Ca²⁺‐programmed stomatal closure induced by experimentally imposed Ca²⁺ oscillations without affecting short‐term Ca²⁺‐reactive closure, mimicking the guard cell behavior of the AtGLR3.1 over‐expressing plants. Our results suggest that over‐expression of AtGLR3.1 impairs Ca²⁺ oscillation‐regulated stomatal movements, and that de novo protein synthesis contributes to the maintenance of long‐term Ca²⁺‐programmed stomatal closure.     

Activation of pre-synaptic M-type K+ channels inhibits [3H]d-aspartate release by reducing Ca2+ entry through P/Q-type voltage-gated Ca2+channels

In this study, the functional consequences of the pharmacological modulation of the M‐current (I_KM) on cytoplasmic Ca²⁺ intracellular Ca²⁺concentration ([Ca²⁺]_i) changes and excitatory neurotransmitter release triggered by various stimuli from isolated rat cortical synaptosomes have been investigated. K_v7.2 immunoreactivity was identified in pre‐synaptic elements in cortical slices and isolated glutamatergic cortical synaptosomes. In cerebrocortical synaptosomes exposed to 20 mM [K⁺]_e, the I_KM activator retigabine (RT, 10 μM) inhibited [³H]d ‐aspartate ([³H]d ‐Asp) release and caused membrane hyperpolarization; both these effects were prevented by the I_KM blocker XE‐991 (20 μM). The I_KM activators RT (0.1–30 μM), flupirtine (10 μM) and BMS‐204352 (10 μM) inhibited 20 mM [K⁺]_e‐induced synaptosomal [Ca²⁺]_i increases; XE‐991 (20 μM) abolished RT‐induced inhibition of depolarization‐triggered [Ca²⁺]_i transients. The P/Q‐type voltage‐sensitive Ca²⁺channel (VSCC) blocker ω‐agatoxin IVA prevented RT‐induced inhibition of depolarization‐induced [Ca²⁺]_i increase and [³H]d ‐Asp release, whereas the N‐type blocker ω‐conotoxin GVIA failed to do so. Finally, 10 μM RT did not modify the increase of [Ca²⁺]_i and the resulting enhancement of [³H]d ‐Asp release induced by [Ca²⁺]_i mobilization from intracellular stores, or by store‐operated Ca²⁺channel activation. Collectively, the present data reveal that the pharmacological activation of I_KM regulates depolarization‐induced [³H]d ‐Asp release from cerebrocortical synaptosomes by selectively controlling the changes of [Ca²⁺]_i occurring through P/Q‐type VSCCs.     

Cytosolic alkalinization is a common and early messenger preceding the production of ROS and NO during stomatal closure by variable signals,including abscisic acid,methyl jasmonate and chitosan

Stomata are unique that they sense and respond to several internal and external stimuli, by modulating signaling components in guard cells. The levels of reactive oxygen species (ROS), nitric oxide (NO) and cytosolic calcium (Ca²⁺) increase significantly during stomatal closure by not only plant hormones [such as abscisic acid (ABA) or methyl jasmonate (MJ)] but also elicitors (such as chitosan). We observed that cytosolic alkalinization preceded the production of ROS as well as NO during ABA induced stomatal closure. We therefore propose that besides ROS and NO, the cytosolic pH is an important secondary messenger during stomatal closure by ABA or MJ. We also noticed that there is either a cross talk or feedback regulation by cytosolic Ca²⁺ and ROS (mostly H₂O₂). Further experiments on the interactions between cytosolic pH, ROS, NO and Ca²⁺ would yield interesting results.Key words: abscisic acid, methyl jasmonate, chitosan, cytosolic pH, reactive oxygen species, H₂O₂, nitric oxide, cytosolic calcium     

Mobilization of Inositol 1,4,5-Trisphosphate-Sensitive Ca2+ Stores Supports Bradykinin- and Muscarinic-Evoked Release of [3H]Noradrenaline from SH-SY5Y Cells

Abstract: The human neuroblastoma cell line SH-SY5Y, maintained at confluence for 14 days, released [³H]-noradrenaline ([³H]NA) when stimulated with either the muscarinic receptor agonist methacholine or bradykinin. The major fraction of release was rapid, occurring in <10 s, whereas nicotine-evoked release was slower. When the extracellular [Ca²⁺] ([Ca²⁺]_e) was buffered to ～50–100 nM, release evoked by nicotine was abolished, whereas that in response to methacholine or bradykinin was reduced by ～50% with EC₅₀ values of ?5.46 ± 0.05 M and ?7.46 ± 0.06 M (log₁₀), respectively. Methacholine and bradykinin also produced rapid elevations of both inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] and intracellular free [Ca²⁺] ([Ca²⁺]_i). These elevations were reduced at low [Ca²⁺]_e and under these conditions the EC₅₀ values for peak elevation of [Ca²⁺]_i were ?6.00 ± 0.14 M for methacholine and ?7.95 ± 0.34 M for bradykinin (n = 3 for all EC₅₀ determinations). At low [Ca²⁺]_e, depletion of nonmitochondrial intracellular Ca²⁺ stores with the Ca²⁺-ATPase inhibitor thapsigargin produced a transient small elevation of [Ca²⁺]_i and a minor release of [³H]NA. At low [Ca²⁺]_e, thapsigargin abolished elevation of [Ca²⁺]_i in response to methacholine and bradykinin and completely inhibited their stimulation of [³H]NA release. It is proposed, therefore, that Ca²⁺ release from Ins(1,4,5)P₃-sensitive stores is a major trigger of methacholine- and bradykinin-evoked [³H]NA release in SH-SY5Y cells.     

Transitional Changes in Membrane Potential and Intracellular [Ca2+] in Rat Basophilic Leukemia Cells

Using whole-cell current-clamp measurements we have found that thapsigargin-mediated activation of store-regulated Ca²⁺ entry in rat basophilic leukemia cells is accompanied by complex changes in membrane potential. These changes consisted of: (i) an initial slow, small depolarization, (ii) a transitional change in potential to a depolarized value and (iii) transitional changes between a hyperpolarized and a depolarized potential. These complex changes in potential can be explained by the interaction between the endogenous inwardly rectifying K⁺ conductance and the generation of a small inward current. To investigate the possible influence of these changes of potential on [Ca²⁺] _i , single cell measurements of fura2 fluorescence were undertaken alone or in combination with current-clamp measurements. Thapsigargin-mediated activation of the store-regulated Ca²⁺ entry pathway was accompanied by a marked increase of [Ca²⁺] _i . During this increase, transient, abrupt declines in [Ca²⁺] _i were detected in approximately 60% of the cells investigated. These changes of [Ca²⁺] _i are consistent with the observed changes of membrane potential recorded under current-clamp. Received: 1 December 1998/Revised: 30 March 1999