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

Stomatal pores formed by a pair of guard cells in the leaf epidermis control gas exchange and transpirational water loss. Stomatal closure is mediated by the release of potassium and anions from guard cells. Anion efflux from guard cells involves slow (S‐type) and rapid (R‐type) anion channels. Recently the SLAC1 gene has been shown to encode the slow, voltage‐independent anion channel component in guard cells. In contrast, the R‐type channel still awaits identification. Here, we show that AtALMT12, a member of the aluminum activated malate transporter family in Arabidopsis, represents a guard cell R‐type anion channel. AtALMT12 is highly expressed in guard cells and is targeted to the plasma membrane. Plants lacking AtALMT12 are impaired in dark‐ and CO₂‐induced stomatal closure, as well as in response to the drought‐stress hormone abscisic acid. Patch‐clamp studies on guard cell protoplasts isolated from atalmt12 mutants revealed reduced R‐type currents compared with wild‐type plants when malate is present in the bath media. Following expression of AtALMT12 in Xenopus oocytes, voltage‐dependent anion currents reminiscent to R‐type channels could be activated. In line with the features of the R‐type channel, the activity of heterologously expressed AtALMT12 depends on extracellular malate. Thereby this key metabolite and osmolite of guard cells shifts the threshold for voltage activation of AtALMT12 towards more hyperpolarized potentials. R‐Type channels, like voltage‐dependent cation channels in nerve cells, are capable of transiently depolarizing guard cells, and thus could trigger membrane potential oscillations, action potentials and initiate long‐term anion and K⁺ efflux via SLAC1 and GORK, respectively.     

Cell type-specific proteomics uncovers a RAF15-SnRK2.6/OST1 kinase cascade in guard cells

Multicellular organisms such as plants contain various cell types with specialized functions. Analyzing the characteristics of each cell type reveals specific cell functions and enhances our understanding of organization and function at the organismal level. Guard cells (GCs) are specialized epidermal cells that regulate the movement of the stomata and gaseous exchange, and provide a model genetic system for analyzing cell fate, signaling, and function. Several proteomics analyses of GC are available, but these are limited in depth. Here we used enzymatic isolation and flow cytometry to enrich GC and mesophyll cell protoplasts and perform in-depth proteomics in these two major cell types in Arabidopsis leaves. We identified approximately 3,000 proteins not previously found in the GC proteome and more than 600 proteins that may be specific to GC. The depth of our proteomics enabled us to uncover a guard cell-specific kinase cascade whereby Raf15 and Snf1-related kinase2.6 (SnRK2.6)/OST1(open stomata 1) mediate abscisic acid (ABA)-induced stomatal closure. RAF15 directly phosphorylated SnRK2.6/OST1 at the conserved Ser175 residue in its activation loop and was sufficient to reactivate the inactive form of SnRK2.6/OST1. ABA-triggered SnRK2.6/OST1 activation and stomatal closure was impaired in raf15 mutants. We also showed enrichment of enzymes and flavone metabolism in GC, and consistent, dramatic accumulation of flavone metabolites. Our study answers the long-standing question of how ABA activates SnRK2.6/OST1 in GCs and represents a resource potentially providing further insights into the molecular basis of GC and mesophyll cell development, metabolism, structure, and function.     

ABA depolarizes guard cells in intact plants, through a transient activation of R- and S-type anion channels

During drought, the plant hormone abscisic acid (ABA) induces rapid stomatal closure and in turn reduces transpiration. Stomatal closure is accompanied by large ion fluxes across the plasma membrane, carried by K+ and anion channels. We recorded changes in the activity of these channels induced by ABA, for guard cells of intact Vicia faba plants. Guard cells in their natural environment were impaled with double-barrelled electrodes, and ABA was applied via the leaf surface. In 45 out of 85 cells tested, ABA triggered a transient depolarization of the plasma membrane. In these cells, the membrane potential partially recovered in the presence of ABA; however, a full recovery of the membrane potentials was only observed after removal of ABA. Repetitive ABA responses could be evoked in single cells, but the magnitude of the response varied from one hormone application to the other. The transient depolarization correlated with the activation of anion channels, which peaked 5 min after introduction of the stimulus. In guard cells with a moderate increase in plasma membrane conductance (DeltaG < 5 nS), ABA predominantly activated voltage-independent (slow (S)-type) anion channels. During strong responses (DeltaG > 5 nS), however, ABA activated voltage-dependent (rapid (R)-type) in addition to S-type anion channels. We conclude that the combined activation of these two channel types leads to the transient depolarization of guard cells. The nature of this ABA response correlates with the transient extrusion of Cl- from guard cells and a rapid but confined reduction in stomatal aperture.     

The plant innate immunity response in stomatal guard cells invokes G-protein-dependent ion channel regulation

Stomata in the epidermis of terrestrial plants are important for CO2 absorption and transpirational water loss, and are also potential points of entry for pathogens. Stomatal opening and closure are controlled by distinct mechanisms. Arabidopsis stomata have been shown to close in response to bacteria and pathogen-associated molecular patterns (PAMPs) as part of PAMP-triggered immunity (PTI). Here we show that flg22, a PAMP derived from bacterial flagellin, also inhibits light-induced stomatal opening. Consistent with our observations on stomatal opening, flg22 inhibits the inward K+ channels (K+ (in) currents) of guard cells that mediate K+ uptake during stomatal opening. Similar to previously documented K+ current changes triggered by exogenous elevation of H(2)O(2) and nitric oxide (NO), with prolonged duration of flg22 exposure the outward K+ channels (K+ (out) currents) of guard cells are also inhibited. In null mutants of the flg22 receptor, FLS2, flg22 regulation of stomatal opening, K+ (in) currents, and K+ (out) currents is eliminated. flg22 also fails to elicit these responses in null mutants of the sole canonical G-protein alpha subunit, GPA1. The bacterial toxin, coronatine, produced by several pathogenic strains of Pseudomonas syringae, reverses the inhibitory effects of flg22 on both K+ (in) currents and stomatal opening, indicating interplay between plant and pathogen in the regulation of plant ion channels. Thus, the PAMP-triggered stomatal response involves K+ channel regulation, and this regulation is dependent on signaling via cognate PAMP receptors and a heterotrimeric G-protein. These new findings provide insights into the largely elusive signaling process underlying PTI-associated guard cell responses.     

Potassium channel currents in intact stomatal guard cells: rapid enhancement by abscisic acid

Evidence of a role for abscisic acid (ABA) in signalling conditions of water stress and promoting stomatal closure is convincing, but past studies have left few clues as to its molecular mechanism(s) of action; arguments centred on changes in H⁺-pump activity and membrane potential, especially, remain ambiguous without the fundamental support of a rigorous electrophysiological analysis. The present study explores the response to ABA of K⁺ channels at the membrane of intact guard cells ofVicia faba L. Membrane potentials were recorded before and during exposures to ABA, and whole-cell currents were measured at intervals throughout to quantitate the steady-state and time-dependent characteristics of the K⁺ channels. On adding 10 M ABA in the presence of 0.1, 3 or 10 mM extracellular K⁺, the free-running membrane potential (V _m) shifted negative-going (–)4–7 mV in the first 5 min of exposure, with no consistent effect thereafter. Voltage-clamp measurements, however, revealed that the K⁺-channel current rose to between 1.84- and 3.41-fold of the controls in the steady-state with a mean halftime of 1.1 ± 0.1 min. Comparable changes in current return via the leak were also evident and accounted for the minimal response inV _m. Calculated atV _m, the K⁺ currents translated to an average 2.65-fold rise in K⁺ efflux with ABA. Abscisic acid was not observed to alter either K⁺-current activation or deactivation.These results are consistent with an ABA-evoked mobilization of K⁺ channels or channel conductance, rather than a direct effect of the phytohormone on K⁺-channel gating. The data discount notions that large swings in membrane voltage are a prerequisite to controlling guard-cell K⁺ flux. Instead, thev highlight a rise in membranecapacity for K⁺ flux, dependent on concerted modulations of K⁺-channel and leak currents, and sufficiently rapid to account generally for the onset of K⁺ loss from guard cells and stomatal closure in ABA.     

The use of voltage-sensitive dyes to monitor signal-induced changes in membrane potential-ABA triggered membrane depolarization in guard cells

The plant membrane potential reports on the activity of electrogenic plasma membrane transport processes. The membrane potential is widely used to report for early events associated with changes in light regime, hormone action or pathogen attacks. The membrane potentials of guard cells can be precisely measured with microelectrodes, but this technique is not well suited for rapid screens with large sample numbers. To provide the basis for large-scale membrane potential recordings, we took advantage of voltage-sensitive dyes. Using the fluorescent dyes bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC(4)(3)) and the FLIPR Membrane Potential Assay Kit (FMP) dye we followed changes in the membrane potential in guard cells and vacuoles. Based on the fluorescence of DiBAC(4)(3) a method was established for quantification of the membrane potential in guard cell protoplasts which should be considered as an excellent system for high-throughput screening of plant cells. In the absence of abscisic acid (ABA), one-third of the guard cell protoplast population spontaneously oscillated for periods of 5-6 min. Upon application of ABA the hyperpolarized fraction ( approximately 50%) of the guard cell protoplast population depolarized within a few minutes. Membrane potential oscillations were terminated by ABA. Oscillations and ABA responses were found in cell populations with active anion channels. Thus time- and voltage-dependent anion channels likely represent the ABA-sensitive conductance and part of the membrane potential oscillator. The suitability of membrane potential dyes was tested on vacuoles, too. Dye-based vacuolar membrane polarization was monitored upon ATP exposure. We conclude that voltage-sensitive dyes provide an excellent tool for the study of changes in the membrane potential in vacuole as well as guard cell populations.     

The structure of Arabidopsis thaliana OST1 provides insights into the kinase regulation mechanism in response to osmotic stress

SnRK [SNF1 (sucrose non-fermenting-1)-related protein kinase] 2.6 [open stomata 1 (OST1)] is well characterized at molecular and physiological levels to control stomata closure in response to water-deficit stress. OST1 is a member of a family of 10 protein kinases from Arabidopsis thaliana (SnRK2) that integrates abscisic acid (ABA)-dependent and ABA-independent signals to coordinate the cell response to osmotic stress. A subgroup of protein phosphatases type 2C binds OST1 and keeps the kinase dephosphorylated and inactive. Activation of OST1 relies on the ABA-dependent inhibition of the protein phosphatases type 2C and the subsequent self-phosphorylation of the kinase. The OST1 ABA-independent activation depends on a short sequence motif that is conserved among all the members of the SnRK2 family. However, little is known about the molecular mechanism underlying this regulation. The crystallographic structure of OST1 shows that ABA-independent regulation motif stabilizes the conformation of the kinase catalytically essential α C helix, and it provides the basis of the ABA-independent regulation mechanism for the SnRK2 family of protein kinases.     

Roles of intracellular hydrogen peroxide accumulation in abscisic acid signaling in Arabidopsis guard cells

Reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂), are among the important second messengers in abscisic acid (ABA) signaling in guard cells. In this study, to investigate specific roles of H₂O₂ in ABA signaling in guard cells, we examined the effects of mutations in the guard cell-expressed catalase (CAT) genes, CAT1 and CAT3, and of the CAT inhibitor 3-aminotriazole (AT) on stomatal movement. The cat3 and cat1 cat3 mutations significantly reduced CAT activities, leading to higher basal level of H₂O₂ in guard cells, when assessed by 2′,7′-dichlorodihydrofluorescein, whereas they did not affect stomatal aperture size under non-stressed condition. In addition, AT-treatment at concentrations that abolish CAT activities, showed trivial affect on stomatal aperture size, while basal H₂O₂ level increased extensively. In contrast, cat mutations and AT-treatment potentiated ABA-induced stomatal closure. Inducible ROS production triggered by ABA was observed in these mutants and wild type as well as in AT-treated guard cells. These results suggest that ABA-inducible cytosolic H₂O₂ elevation functions in ABA-induced stomatal closure, while constitutive increase of H₂O₂ do not cause stomatal closure.     

The role of PLDα1 in providing specificity to signal‐response coupling by heterotrimeric G‐protein components in Arabidopsis

Heterotrimeric G‐proteins comprised of Gα, Gβ and Gγ subunits are important signal transducers in all eukaryotes. In plants, G‐proteins affect multiple biotic and abiotic stress responses, as well as many developmental processes, even though their repertoire is significantly limited compared with that in metazoan systems. One canonical and three extra‐large Gα, 1 Gβ and 3 Gγ proteins represent the heterotrimeric G‐protein complex in Arabidopsis, and a single regulatory protein, RGS1, is one of the few known biochemical regulators of this signaling complex. This quantitative disparity between the number of signaling components and the range of processes they influence is rather intriguing. We now present evidence that the phospholipase Dα1 protein is a key component and modulator of the G‐protein complex in affecting a subset of signaling pathways. We also show that the same G‐protein subunits and their modulators exhibit distinct physiological and genetic interactions depending on specific signaling and developmental pathways. Such developmental plasticity and interaction specificity likely compensates for the lack of multiplicity of individual subunits, and helps to fine tune the plants' responses to constantly changing environments.     

Ins(1,4,5)P3 receptor type 1 associates with AKAP9 (AKAP450 variant) and protein kinase A type IIβ in the Golgi apparatus in cerebellar granule cells

Background information. Interconnections between the Ca²⁺ and cAMP signalling pathways can determine the specificity and diversity of the cellular effects mediated by these second messengers. Most cAMP effects are mediated by PKA (protein kinase A), which is anchored close to its membranous substrates by AKAPs (A kinase‐anchoring proteins). In many cell types, the activation of InsP₃R (inositol 1,4,5‐trisphosphate receptor), an endoplasmic reticulum Ca²⁺ channel, is a key event of Ca²⁺ signalling. The phosphorylation of InsP₃R1 by PKA stimulates Ca²⁺ mobilization. This control is thought to be tight, involving the association of PKA with InsP₃R1. The InsP₃R1 isoform predominates in central nervous tissue and its concentration is highest in the cerebellar microsomes. We investigated the complex formed by InsP₃R1 and PKA in this fraction, vith a view to identifying its components and determining its distribution in the cerebellar cortex. Results. Immunoprecipitation experiments showed that InsP₃R1 associated with PKA type IIβ and AKAP450, the longer variant of AKAP9, in sheep cerebellar microsomes. The co‐purification of AKAP450 with InsP₃R1 on heparin‐agarose provided further evidence of the association of these proteins. Immunohistofluorescence experiments on slices of cerebellar cortex showed that AKAP450 was colocalized with InsP₃R1 and RIIβ (regulatory subunit of PKA IIβ) in granule cells, but not in Purkinje cells. AKAP450 was localized in the Golgi apparatus of these two cell types whereas InsP₃R1 was detected in this organelle only in granule cells. Conclusions. Taken together these results suggest that InsP₃R1 forms a complex with AKAP450 and PKAIIβ, localized in the Golgi apparatus of cerebellar granule cells. In contrast, the association of InsP₃R1 with PKA in Purkinje cells would require a different macromolecular complex.     

JNK-dependent NFATc1 pathway positively regulates IL-13 gene expression induced by (–)-epigallocatechin-3-gallate in human basophilic KU812 cells

(–)-Epigallocatechin-3-gallate (EGCG) has been reported to possess a wide range of biological and pharmacological properties. In this study, we investigated the effects of EGCG on IL-13 gene expression in human basophilic KU812 cells. The IL-13 mRNA expression level was dose-dependently increased by treatment with EGCG (5–20 μM) for 1 h and additional incubation in a medium for 23 h. EGCG significantly increased the intracellular peroxide level as detected by the peroxide-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate. A pharmacological experiment using catalase and a structure–activity relationship study revealed that the exogenously produced H₂O₂ significantly, but partially, contributed to the IL-13 expression as well as the intracellular oxidative status. Furthermore, EGCG at the concentration required for IL-13 up-regulation activated c-Jun NH₂-terminal kinase (JNK), but not extracellular signal-regulated protein kinase or p38 mitogen-activated protein kinase in KU812 cells. Transfection of a JNK-specific siRNA as well as treatment with a JNK-specific inhibitor, SP600125, significantly reduced the EGCG-induced IL-13 mRNA expression, by 47.1 and 44.6%, respectively. In addition, we observed the nuclear translocation, mRNA up-regulation, and activation of DNA binding with the IL-13 promoter of nuclear factor of activated T cells (NFATc1) in the EGCG-treated cells. These data provide biological evidence that EGCG induces IL-13 mRNA expression via the JNK-dependent NFATc1 pathway in KU812 cells.