Activation Of S-type Anion Channels Research Articles

Calcium Channels, OST1 and Stomatal Defence: Current Status and Beyond

Stomatal immunity is regulated by pathogen-associated molecular patterns (PAMPs)- and abscisic acid (ABA)-triggered signalling in different ways. Cytoplasmic Ca2+ signature in the guard cells plays a vital function in stomatal immunity, but the mechanism of Ca2+ import is unknown. It has been very recently established that the hyperosmolality-gated calcium-permeable channels (OSCAs) and cyclic nucleotide-gated channels (CNGCs) are responsible for the influx of Ca2+ in the cytoplasm, which are activated after BIK1-mediated phosphorylation and ABA interaction during PAMPs- and ABA-triggered stomatal immunity in plants, respectively. Further, ABA-triggered OPEN STOMATA1 (OST1) causes the disassembly of microtubules in the guard cells besides activation of S-type anion channels (SLAC1) for the efflux of cytoplasmic anions that leads to stomata closure.

Stomata: the holey grail of plant evolution

Stomata: the holey grail of plant evolution

Ethylene Inhibits Methyl Jasmonate-Induced Stomatal Closure by Modulating Guard Cell Slow-Type Anion Channel Activity via the OPEN STOMATA 1/SnRK2.6 Kinase-Independent Pathway in Arabidopsis.

Signal crosstalk between jasmonate and ethylene is crucial for a proper maintenance of defense responses and development. Although previous studies reported that both jasmonate and ethylene also function as modulators of stomatal movements, the signal crosstalk mechanism in stomatal guard cells remains unclear. Here, we show that the ethylene signaling inhibits jasmonate signaling as well as abscisic acid (ABA) signaling in guard cells of Arabidopsis thaliana and reveal the signaling crosstalk mechanism. Both an ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and an ethylene-releasing compound ethephon induced transient stomatal closure, and also inhibited methyl jasmonate (MeJA)-induced stomatal closure as well as ABA-induced stomatal closure. The ethylene inhibition of MeJA-induced stomatal closure was abolished in the ethylene-insensitive mutant etr1-1, whereas MeJA-induced stomatal closure was impaired in the ethylene-overproducing mutant eto1-1. Pretreatment with ACC inhibited MeJA-induced reactive oxygen species (ROS) production as well as ABA-induced ROS production in guard cells but did not suppress ABA activation of OPEN STOMATA 1 (OST1) kinase in guard cell-enriched epidermal peels. The whole-cell patch-clamp analysis revealed that ACC attenuated MeJA and ABA activation of S-type anion channels in guard cell protoplasts. However, MeJA and ABA inhibitions of Kin channels were not affected by ACC pretreatment. These results suggest that ethylene signaling inhibits MeJA signaling and ABA signaling by targeting S-type anion channels and ROS but not OST1 kinase and K+ channels in Arabidopsis guard cells.

Mitogen-activated protein kinases MPK4 and MPK12 are key components mediating CO2 -induced stomatal movements.

Respiration in leaves and the continued elevation in the atmospheric CO2 concentration cause CO2 -mediated reduction in stomatal pore apertures. Several mutants have been isolated for which stomatal responses to both abscisic acid (ABA) and CO2 are simultaneously defective. However, there are only few mutations that impair the stomatal response to elevated CO2 , but not to ABA. Such mutants are invaluable in unraveling the molecular mechanisms of early CO2 signal transduction in guard cells. Recently, mutations in the mitogen-activated protein (MAP) kinase, MPK12, have been shown to partially impair CO2 -induced stomatal closure. Here, we show that mpk12 plants, in which MPK4 is stably silenced specifically in guard cells (mpk12 mpk4GC homozygous double-mutants), completely lack CO2 -induced stomatal responses and have impaired activation of guard cell S-type anion channels in response to elevated CO2 /bicarbonate. However, ABA-induced stomatal closure, S-type anion channel activation and ABA-induced marker gene expression remain intact in the mpk12 mpk4GC double-mutants. These findings suggest that MPK12 and MPK4 act very early in CO2 signaling, upstream of, or parallel to the convergence of CO2 and ABA signal transduction. The activities of MPK4 and MPK12 protein kinases were not directly modulated by CO2 /bicarbonate invitro, suggesting that they are not direct CO2 /bicarbonate sensors. Further data indicate that MPK4 and MPK12 have distinguishable roles in Arabidopsis and that the previously suggested role of RHC1 in stomatal CO2 signaling is minor, whereas MPK4 and MPK12 act as key components of early stomatal CO2 signal transduction.

Identification of SLAC1 anion channel residues required for CO2/bicarbonate sensing and regulation of stomatal movements

Increases in CO2 concentration in plant leaves due to respiration in the dark and the continuing atmospheric [CO2] rise cause closing of stomatal pores, thus affecting plant-water relations globally. However, the underlying CO2/bicarbonate (CO2/HCO3-) sensing mechanisms remain unknown. [CO2] elevation in leaves triggers stomatal closure by anion efflux mediated via the SLAC1 anion channel localized in the plasma membrane of guard cells. Previous reconstitution analysis has suggested that intracellular bicarbonate ions might directly up-regulate SLAC1 channel activity. However, whether such a CO2/HCO3- regulation of SLAC1 is relevant for CO2 control of stomatal movements in planta remains unknown. Here, we computationally probe for candidate bicarbonate-interacting sites within the SLAC1 anion channel via long-timescale Gaussian accelerated molecular dynamics (GaMD) simulations. Mutations of two putative bicarbonate-interacting residues, R256 and R321, impaired the enhancement of the SLAC1 anion channel activity by CO2/HCO3- in Xenopus oocytes. Mutations of the neighboring charged amino acid K255 and residue R432 and the predicted gate residue F450 did not affect HCO3- regulation of SLAC1. Notably, gas-exchange experiments with slac1-transformed plants expressing mutated SLAC1 proteins revealed that the SLAC1 residue R256 is required for CO2 regulation of stomatal movements in planta, but not for abscisic acid (ABA)-induced stomatal closing. Patch clamp analyses of guard cells show that activation of S-type anion channels by CO2/HCO3-, but not by ABA, was impaired, indicating the relevance of R256 for CO2 signal transduction. Together, these analyses suggest that the SLAC1 anion channel is one of the physiologically relevant CO2/HCO3- sensors in guard cells.

Natural Variation in Arabidopsis Cvi-0 Accession Reveals an Important Role of MPK12 in Guard Cell CO2 Signaling.

Plant gas exchange is regulated by guard cells that form stomatal pores. Stomatal adjustments are crucial for plant survival; they regulate uptake of CO2 for photosynthesis, loss of water, and entrance of air pollutants such as ozone. We mapped ozone hypersensitivity, more open stomata, and stomatal CO2-insensitivity phenotypes of the Arabidopsis thaliana accession Cvi-0 to a single amino acid substitution in MITOGEN-ACTIVATED PROTEIN (MAP) KINASE 12 (MPK12). In parallel, we showed that stomatal CO2-insensitivity phenotypes of a mutant cis (CO2-insensitive) were caused by a deletion of MPK12. Lack of MPK12 impaired bicarbonate-induced activation of S-type anion channels. We demonstrated that MPK12 interacted with the protein kinase HIGH LEAF TEMPERATURE 1 (HT1)—a central node in guard cell CO2 signaling—and that MPK12 functions as an inhibitor of HT1. These data provide a new function for plant MPKs as protein kinase inhibitors and suggest a mechanism through which guard cell CO2 signaling controls plant water management.

Calcium specificity signaling mechanisms in abscisic acid signal transduction in Arabidopsis guard cells.

A central question is how specificity in cellular responses to the eukaryotic second messenger Ca(2+) is achieved. Plant guard cells, that form stomatal pores for gas exchange, provide a powerful system for in depth investigation of Ca(2+)-signaling specificity in plants. In intact guard cells, abscisic acid (ABA) enhances (primes) the Ca(2+)-sensitivity of downstream signaling events that result in activation of S-type anion channels during stomatal closure, providing a specificity mechanism in Ca(2+)-signaling. However, the underlying genetic and biochemical mechanisms remain unknown. Here we show impairment of ABA signal transduction in stomata of calcium-dependent protein kinase quadruple mutant plants. Interestingly, protein phosphatase 2Cs prevent non-specific Ca(2+)-signaling. Moreover, we demonstrate an unexpected interdependence of the Ca(2+)-dependent and Ca(2+)-independent ABA-signaling branches and the in planta requirement of simultaneous phosphorylation at two key phosphorylation sites in SLAC1. We identify novel mechanisms ensuring specificity and robustness within stomatal Ca(2+)-signaling on a cellular, genetic, and biochemical level.

Open Stomata 1 Kinase is Essential for Yeast Elicitor-Induced Stomatal Closure in Arabidopsis.

We recently demonstrated that yeast elicitor (YEL)-induced stomatal closure requires a Ca(2+)-dependent kinase, CPK6. A Ca(2+)-independent kinase, Open Stomata 1 (OST1), is involved in stomatal closure induced by various stimuli including ABA. In the present study, we investigated the role of OST1 in YEL-induced stomatal closure in Arabidopsis using a knock-out mutant, ost1-3, and a kinase-deficient mutant, ost1-2. YEL did not induce stomatal closure or activation of guard cell S-type anion channels in the ost1 mutants unlike in wild-type plants. However, YEL did not increase OST1 kinase activity in wild-type guard cells. The YEL-induced stomatal closure and activation of S-type anion channels were also impaired in a gain-of-function mutant of a clade A type 2C protein phosphatase (ABA INSENSITIVE 1), abi1-1C. In the ost1 mutants like in the wild type, YEL induced H2O2 accumulation, activation of non-selective Ca(2+)-permeable cation (ICa) channels and transient elevations in cytosolic free Ca(2+) concentration ([Ca(2+)]cyt) in guard cells. These results suggest that OST1 kinase is essential for stomatal closure and activation of S-type anion channels induced by YEL and that OST1 is not involved in H2O2 accumulation, ICa channel activation or [Ca(2+)]cyt elevations in guard cells induced by YEL.

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 Ca(2+) concentration ([Ca(2+)]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 [Ca(2+)]cyt elevation in guard cell MeJA signalling, suggesting that MPK9 and MPK12 are key regulators mediating both ABA and MeJA signalling in guard cells.

PYR/RCAR Receptors Contribute to Ozone-, Reduced Air Humidity-, Darkness-, and CO2-Induced Stomatal Regulation

Rapid stomatal closure induced by changes in the environment, such as elevation of CO2, reduction of air humidity, darkness, and pulses of the air pollutant ozone (O3), involves the SLOW ANION CHANNEL1 (SLAC1). SLAC1 is activated by OPEN STOMATA1 (OST1) and Ca(2+)-dependent protein kinases. OST1 activation is controlled through abscisic acid (ABA)-induced inhibition of type 2 protein phosphatases (PP2C) by PYRABACTIN RESISTANCE/REGULATORY COMPONENTS OF ABA RECEPTOR (PYR/RCAR) receptor proteins. To address the role of signaling through PYR/RCARs for whole-plant steady-state stomatal conductance and stomatal closure induced by environmental factors, we used a set of Arabidopsis (Arabidopsis thaliana) mutants defective in ABA metabolism/signaling. The stomatal conductance values varied severalfold among the studied mutants, indicating that basal ABA signaling through PYR/RCAR receptors plays a fundamental role in controlling whole-plant water loss through stomata. PYR/RCAR-dependent inhibition of PP2Cs was clearly required for rapid stomatal regulation in response to darkness, reduced air humidity, and O3. Furthermore, PYR/RCAR proteins seem to function in a dose-dependent manner, and there is a functional diversity among them. Although a rapid stomatal response to elevated CO2 was evident in all but slac1 and ost1 mutants, the bicarbonate-induced activation of S-type anion channels was reduced in the dominant active PP2C mutants abi1-1 and abi2-1. Further experiments with a wider range of CO2 concentrations and analyses of stomatal response kinetics suggested that the ABA signalosome partially affects the CO2-induced stomatal response. Thus, we show that PYR/RCAR receptors play an important role for the whole-plant stomatal adjustments and responses to low humidity, darkness, and O3 and are involved in responses to elevated CO2.

Barley mildew and its elicitor chitosan promote closed stomata by stimulating guard‐cell S‐type anion channels

Stomatal closure is known to be associated with early defence responses of plant cells triggered by microbe-associated molecular patterns (MAMPs). However, the molecular mechanisms underlying these guard-cell responses have not yet been elucidated. We therefore studied pathogen-induced changes in ion channel activity in Hordeum vulgare guard cells. Barley mildew (Blumeria graminis) hyphae growing on leaves inhibited light-induced stomatal opening, starting at 9 h after inoculation, when appressoria had developed. Alternatively, stomatal closure was induced by nano-infusion of chitosan via open stomata into the sub-stomatal cavity. Experiments using intracellular double-barreled micro-electrodes revealed that mildew stimulated S-type (slow) anion channels in guard cells. These channels enable the efflux of anions from guard cells and also promote K(+) extrusion by altering the plasma membrane potential. Stimulation of S-type anion channels was also provoked by nano-infusion of chitosan. These data suggest that MAMPs of mildew hyphae penetrating the cuticle provoke activation of S-type anion channels in guard cells. In response, guard cells extrude K(+) salts, resulting in stomatal closure. Plasma membrane anion channels probably represent general targets of MAMP signaling in plants, as these elicitors depolarize the plasma membrane of various cell types.

Central functions of bicarbonate in S-type anion channel activation and OST1 protein kinase in CO2 signal transduction in guard cell.

Plants respond to elevated CO(2) via carbonic anhydrases that mediate stomatal closing, but little is known about the early signalling mechanisms following the initial CO(2) response. It remains unclear whether CO(2), HCO(3)(-) or a combination activates downstream signalling. Here, we demonstrate that bicarbonate functions as a small-molecule activator of SLAC1 anion channels in guard cells. Elevated intracellular [HCO(3)(-)](i) with low [CO(2)] and [H(+)] activated S-type anion currents, whereas low [HCO(3)(-)](i) at high [CO(2)] and [H(+)] did not. Bicarbonate enhanced the intracellular Ca(2+) sensitivity of S-type anion channel activation in wild-type and ht1-2 kinase mutant guard cells. ht1-2 mutant guard cells exhibited enhanced bicarbonate sensitivity of S-type anion channel activation. The OST1 protein kinase has been reported not to affect CO(2) signalling. Unexpectedly, OST1 loss-of-function alleles showed strongly impaired CO(2)-induced stomatal closing and HCO(3)(-) activation of anion channels. Moreover, PYR/RCAR abscisic acid (ABA) receptor mutants slowed but did not abolish CO(2)/HCO(3)(-) signalling, redefining the convergence point of CO(2) and ABA signalling. A new working model of the sequence of CO(2) signalling events in gas exchange regulation is presented.

Ca2+-dependent activation of guard cell anion channels, triggered by hyperpolarization, is promoted by prolonged depolarization

Rapid stomatal closure is driven by the activation of S-type anion channels in the plasma membrane of guard cells. This response has been linked to Ca(2+) signalling, but the impact of transient Ca(2+) signals on S-type anion channel activity remains unknown. In this study, transient elevation of the cytosolic Ca(2+) level was provoked by voltage steps in guard cells of intact Nicotiana tabacum plants. Changes in the activity of S-type anion channels were monitored using intracellular triple-barrelled micro-electrodes. In cells kept at a holding potential of -100 mV, voltage steps to -180 mV triggered elevation of the cytosolic free Ca(2+) concentration. The increase in the cytosolic Ca(2+) level was accompanied by activation of S-type anion channels. Guard cell anion channels were activated by Ca(2+) with a half maximum concentration of 515 nm (SE = 235) and a mean saturation value of -349 pA (SE = 107) at -100 mV. Ca(2+) signals could also be evoked by prolonged (100 sec) depolarization of the plasma membrane to 0 mV. Upon returning to -100 mV, a transient increase in the cytosolic Ca(2+) level was observed, activating S-type channels without measurable delay. These data show that cytosolic Ca(2+) elevation can activate S-type anion channels in intact guard cells through a fast signalling pathway. Furthermore, prolonged depolarization to 0 mV alters the activity of Ca(2+) transport proteins, resulting in an overshoot of the cytosolic Ca(2+) level after returning the membrane potential to -100 mV.

Roles of AtTPC1, Vacuolar Two Pore Channel 1, in Arabidopsis Stomatal Closure

Abscisic acid (ABA) induces production of reactive oxygen species (ROS) and nitric oxide (NO), elevation of the cytosolic free calcium ion concentration ([Ca(2+)](cyt)) and cytosolic pH (pH(cyt)), and activation of S-type anion channels in guard cells, causing stomatal closure. To investigate whether Arabidopsis Two pore channel 1 (AtTPC1) that encodes the slow vacuolar (SV) channel is involved in stomatal closure, we examined stomatal movements and mobilization of second messengers in the attpc1-2 loss-of-function mutant in response to ABA, methyl jasmonate (MeJA) and Ca(2+). Both ABA and MeJA elicited production of ROS and NO, [Ca(2+)](cyt) oscillations, cytosolic alkalization and activation of S-type anion channel currents to lead to stomatal closure in the attpc1-2 mutant as well as the wild type. Unlike the wild type, in the attpc1-2 mutant exogenous Ca(2+) neither induced stomatal closure nor activated plasma membrane S-type anion channel currents despite [Ca(2+)](cyt) elevation. These results indicate that AtTPC1 functions in response to external Ca(2+) but not to ABA and MeJA in Arabidopsis guard cells and suggest that AtTPC1 could be involved in priming of plasma membrane S-type anion channels by external Ca(2+) in Arabidopsis guard cells.

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

Cytosolic Ca(2+) ([Ca(2+)](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, [Ca(2+)](cyt) has been shown to regulate stomatal movements, and a defined window of Ca(2+) oscillation kinetic parameters encodes necessary information for long-term stomatal movements. However, it remains unknown how the encrypted information in the cytosolic Ca(2+) 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 Ca(2+)-induced stomatal closure. Cytosolic Ca(2+) activation of S-type anion channels, which play a central role in Ca(2+)-reactive stomatal closure, was normal in the AtGLR3.1 over-expressing plants. Interestingly, AtGLR3.1 over-expression did not affect Ca(2+)-induced Ca(2+) oscillation kinetics, but resulted in a failure to maintain long-term 'Ca(2+)-programmed' stomatal closure when Ca(2+) oscillations containing information for maintaining stomatal closure were imposed. By contrast, prompt short-term Ca(2+)-reactive closure was not affected in AtGLR3.1 over-expressing plants. In wild-type plants, the translational inhibitor cyclohexamide partially inhibited Ca(2+)-programmed stomatal closure induced by experimentally imposed Ca(2+) oscillations without affecting short-term Ca(2+)-reactive closure, mimicking the guard cell behavior of the AtGLR3.1 over-expressing plants. Our results suggest that over-expression of AtGLR3.1 impairs Ca(2+) oscillation-regulated stomatal movements, and that de novo protein synthesis contributes to the maintenance of long-term Ca(2+)-programmed stomatal closure.

Two types of anion channel currents in guard cells with distinct voltage regulation.

Transpirational water loss by plants is reduced by closing of stomatal pores in the leaf epidermis. Anion channels in the plasma membrane of guard cells may provide a key molecular mechanism for control of stomatal closing in leaves. However, central questions regarding the regulation, diversity, and function of anion channels in guard cells and other higher plant cells remain unanswered. We show here that two highly distinct types of depolarization-activated anion currents operate in the plasma membrane of Vicia faba guard cells. One described type of anion channel was activated rapidly within 50 ms by depolarization, inactivated during prolonged stimulation, and deactivated rapidly at hyperpolarized potentials (R-type anion current). The other depolarization-activated anion current showed extremely slow voltage-dependent activation and deactivation (S-type anion current) and lacked inactivation. The distinct voltage and time dependencies of R-type and S-type anion channels suggest that they may play a role during depolarization-associated signal transduction in higher plant cells and that these anion channels may contribute to different processes in the regulation of stomatal movements. In particular, the slow and sustained nature of S-type anion channel activation revealed here leads us to hypothesize that S-type anion channels may provide a central molecular mechanism for control of stomatal closing, which is accompanied by long-term anion efflux and depolarization.