Nitric oxide regulates K+ and Cl- channels in guard cells through a subset of abscisic acid-evoked signaling pathways

Abscisic acid (ABA) triggers a complex sequence of signaling events that lead to concerted modulation of ion channels at the plasma membrane of guard cells and solute efflux to drive stomatal closure in plant leaves. Recent work has indicated that nitric oxide (NO) and its synthesis are a prerequisite for ABA signal transduction in Arabidopsis and Vicia guard cells. Its mechanism(s) of action is not well defined in guard cells and, generally, in higher plants. Here we show directly that NO selectively regulates Ca2+-sensitive ion channels of Vicia guard cells by promoting Ca2+ release from intracellular stores to raise cytosolic-free [Ca2+]. NO-sensitive Ca2+ release was blocked by antagonists of guanylate cyclase and cyclic ADP ribose-dependent endomembrane Ca2+ channels, implying an action mediated via a cGMP-dependent cascade. NO did not recapitulate ABA-evoked control of plasma membrane Ca2+ channels and Ca2+-insensitive K+ channels, and NO scavengers failed to block the activation of these K+ channels evoked by ABA. These results place NO action firmly within one branch of the Ca2+-signaling pathways engaged by ABA and define the boundaries of parallel signaling events in the control of guard cell movements.     

Nitric oxide regulates endocytosis by S-nitrosylation of dynamin

The GTPase dynamin regulates endocytic vesicle budding from the plasma membrane, but the molecular mechanisms involved remain incompletely understood. We report that dynamin, which interacts with NO synthase, is S-nitrosylated at a single cysteine residue (C607) after stimulation of the beta(2) adrenergic receptor. S-nitrosylation increases dynamin self-assembly and GTPase activity and facilitates its redistribution to the membrane. A mutant protein bearing a C607A substitution does not self-assemble properly or increase its enzymatic activity in response to NO. In NO-generating cells, expression of dynamin C607A, like the GTPase-deficient dominant-negative K44A dynamin, inhibits both beta(2) adrenergic receptor internalization and bacterial invasion. Furthermore, exogenous or endogenously produced NO enhances internalization of both beta(2) adrenergic and epidermal growth factor receptors. Thus, NO regulates endocytic vesicle budding by S-nitrosylation of dynamin. Collectively, our data suggest a general NO-dependent mechanism by which the trafficking of receptors may be regulated and raise the idea that pathogenic microbes and viruses may induce S-nitrosylation of dynamin to facilitate cellular entry.     

Nitric oxide negatively regulates c-Jun N-terminal kinase/stress-activated protein kinase by means of S-nitrosylation

NO, produced from l-arginine in a reaction catalyzed by NO synthase, is an endogenous free radical with multiple functions in mammalian cells. Here, we demonstrate that endogenously produced NO can suppress c-Jun N-terminal kinase (JNK) activation in intact cells. Treatment of BV-2 murine microglial cells with IFN-gamma induced endogenous NO production, concomitantly suppressing JNK1 activation. Similarly, IFN-gamma induced suppression of JNK1 activation in RAW264.7 murine macrophage cells and rat alveolar macrophages. The IFN-gamma-induced suppression of JNK1 activation in BV-2, RAW264.7, or rat alveolar macrophage cells was completely prevented by N(G)-nitro-l-arginine, a NO synthase inhibitor. Interestingly, the IFN-gamma-induced suppression of JNK1 activation was not affected by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of guanylyl cyclase. 8-Bromo-cGMP, a membrane-permeant analogue of cGMP, did not change JNK1 activation in intact cells either. In contrast, S-nitro-N-acetyl-dl-penicillamine (SNAP), a NO donor, inhibited JNK1 activity in vitro. Furthermore, a thiol reducing agent, DTT, reversed not only the in vitro inhibition of JNK1 activity by SNAP but also the in vivo suppression of JNK1 activity by IFN-gamma. Substitution of serine for cysteine-116 in JNK1 abolished the inhibitory effect of IFN-gamma or SNAP on JNK1 activity in vivo or in vitro, respectively. Moreover, IFN-gamma enhanced endogenous S-nitrosylation of JNK1 in RAW264.7 cells. Collectively, our data suggest that endogenous NO mediates the IFN-gamma-induced suppression of JNK1 activation in macrophage cells by means of a thiol-redox mechanism.     

Strong regulation of slow anion channels and abscisic acid signaling in guard cells by phosphorylation and dephosphorylation events

Recent evidence suggests that slow anion channels in guard cells need to be activated to trigger stomatal closing and efficiently inactivated during stomatal opening. The patch-clamp technique was employed here to determine mechanisms that produce strong regulation of slow anion channels in guard cells. MgATP in guard cells, serving as a donor for phosphorylation, leads to strong activation of slow anion channels. Slow anion-channel activity was almost completely abolished by removal of cytosolic ATP or by the kinase inhibitors K-252a and H7. Nonhydrolyzable ATP, GTP, and guanosine 5'-[gamma-thio]triphosphate did not replace the ATP requirement for anion-channel activation. In addition, down-regulation of slow anion channels by ATP removal was inhibited by the phosphatase inhibitor okadaic acid. Stomatal closures in leaves induced by the plant hormone abscisic acid (ABA) and malate were abolished by kinase inhibitors and/or enhanced by okadaic acid. These data suggest that ABA signal transduction may proceed by activation of protein kinases and inhibition of an okadaic acid-sensitive phosphatase. This modulation of ABA-induced stomatal closing correlated to the large dynamic range for up- and down-regulation of slow anion channels by opposing phosphorylation and dephosphorylation events in guard cells. The presented opposing regulation by kinase and phosphatase modulators could provide important mechanisms for signal transduction by ABA and other stimuli during stomatal movements.     

Nitric oxide suppresses NADPH oxidase-dependent superoxide production by S-nitrosylation in human endothelial cells

OBJECTIVE: Endothelial NADPH oxidase is a major source of superoxide in blood vessels and is implicated in the oxidative stress accompanying vascular diseases, including atherosclerosis. Here we investigate the regulation of NADPH oxidase activity by nitric oxide (NO). METHODS: Human cultured microvascular endothelial cells (HMEC-1) were treated with the NO donors, diethylenetriamine (DETA)-NONOate, S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) for 0.5-24 h. Superoxide production was measured by lucigenin chemiluminescence and dihydroethidium fluorescence, while NADPH oxidase subunit expression was measured via Western blotting. S-nitrosylation was assessed using the 2,3-diaminonapthalene (DAN) assay, and via immunoblotting with an anti-nitrosocysteine antibody. RESULTS: Specific siRNA reduced Nox2 and Nox4 protein expression and markedly decreased superoxide production in HMEC-1. DETA-NONOate (10-300 micromol/L) suppressed superoxide production in HMEC-1 in a concentration- and time-dependent manner, which was not entirely attributable to stoichiometric reaction with NO, for the effect was observed more than 6 h after removing DETA-NONOate from solution. Similarly, sustained attenuation of superoxide production was achieved with SNP (10-100 micromol/L) and SNAP (10-100 micromol/L). The suppressive effect of NO was not dependent on (1) the sGC/cGMP/PKG pathway, (2) peroxynitrite-formation, (3) reduced protein expression of NADPH oxidase subunits or (4) dissociation of NADPH oxidase subunits. Treatment with NO caused S-nitrosylation of the crucial organizer subunit p47phox, and de-nitrosylation with UV light restored superoxide production. CONCLUSIONS: NO causes sustained suppression of NADPH oxidase-dependent superoxide production in human endothelial cells by S-nitrosylation of p47phox. These findings highlight a novel approach by which vascular oxidative stress might be suppressed by NO donors.     

Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair

In response to drought stress the phytohormone ABA (abscisic acid) induces stomatal closure and, therein, activates guard cell anion channels in a calcium-dependent as well as-independent manner. Two key components of the ABA signaling pathway are the protein kinase OST1 (open stomata 1) and the protein phosphatase ABI1 (ABA insensitive 1). The recently identified guard cell anion channel SLAC1 appeared to be the key ion channel in this signaling pathway but remained electrically silent when expressed heterologously. Using split YFP assays, we identified OST1 as an interaction partner of SLAC1 and ABI1. Upon coexpression of SLAC1 with OST1 in Xenopus oocytes, SLAC1-related anion currents appeared similar to those observed in guard cells. Integration of ABI1 into the SLAC1/OST1 complex, however, prevented SLAC1 activation. Our studies demonstrate that SLAC1 represents the slow, deactivating, weak voltage-dependent anion channel of guard cells controlled by phosphorylation/dephosphorylation.     

Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action

The plant hormone abscisic acid (ABA) is produced in response to abiotic stresses and mediates stomatal closure in response to drought via recently identified ABA receptors (pyrabactin resistance/regulatory component of ABA receptor; PYR/RCAR). SLAC1 encodes a central guard cell S-type anion channel that mediates ABA-induced stomatal closure. Coexpression of the calcium-dependent protein kinase 21 (CPK21), CPK23, or the Open Stomata 1 kinase (OST1) activates SLAC1 anion currents. However, reconstitution of ABA activation of any plant ion channel has not yet been attained. Whether the known core ABA signaling components are sufficient for ABA activation of SLAC1 anion channels or whether additional components are required remains unknown. The Ca(2+)-dependent protein kinase CPK6 is known to function in vivo in ABA-induced stomatal closure. Here we show that CPK6 robustly activates SLAC1-mediated currents and phosphorylates the SLAC1 N terminus. A phosphorylation site (S59) in SLAC1, crucial for CPK6 activation, was identified. The group A PP2Cs ABI1, ABI2, and PP2CA down-regulated CPK6-mediated SLAC1 activity in oocytes. Unexpectedly, ABI1 directly dephosphorylated the N terminus of SLAC1, indicating an alternate branched early ABA signaling core in which ABI1 targets SLAC1 directly (down-regulation). Furthermore, here we have successfully reconstituted ABA-induced activation of SLAC1 channels in oocytes using the ABA receptor pyrabactin resistant 1 (PYR1) and PP2C phosphatases with two alternate signaling cores including either CPK6 or OST1. Point mutations in ABI1 disrupting PYR1-ABI1 interaction abolished ABA signal transduction. Moreover, by addition of CPK6, a functional ABA signal transduction core from ABA receptors to ion channel activation was reconstituted without a SnRK2 kinase.     

Endothelial nitric oxide synthase negatively regulates hydrogen peroxide-stimulated AMP-activated protein kinase in endothelial cells

Hydrogen peroxide and other reactive oxygen species are intimately involved in endothelial cell signaling. In many cell types, the AMP-activated protein kinase (AMPK) has been implicated in the control of metabolic responses, but the role of endothelial cell redox signaling in the modulation of AMPK remains to be completely defined. We used RNA interference and pharmacological methods to establish that H₂O₂ is a critical activator of AMPK in cultured bovine aortic endothelial cells (BAECs). H₂O₂ treatment of BAECs rapidly and significantly increases the phosphorylation of AMPK. The EC₅₀ for H₂O₂-promoted phosphorylation of AMPK is 65 ± 15 μM, within the physiological range of cellular H₂O₂ concentrations. The Ca²⁺/calmodulin-dependent protein kinase kinase-β (CaMKKβ) inhibitor STO-609 abolishes H₂O₂-dependent AMPK activation, whereas eNOS inhibitors enhance AMPK activation. Similarly, siRNA-mediated knockdown of CaMKKβ abrogates AMPK activation, whereas siRNA-mediated knockdown of eNOS leads to a striking increase in AMPK phosphorylation. Cellular imaging studies using the H₂O₂ biosensor HyPer show that siRNA-mediated eNOS knockdown leads to a marked increase in intracellular H₂O₂ generation, which is blocked by PEG-catalase. eNOS^−/− mice show a marked increase in AMPK phosphorylation in liver and lung compared to wild-type mice. Lung endothelial cells from eNOS^−/− mice also show a significant increase in AMPK phosphorylation. Taken together, these results establish that CaMKKβ is critically involved in mediating the phosphorylation of AMPK promoted by H₂O₂ in endothelial cells, and document that eNOS is an important negative regulator of AMPK phosphorylation and intracellular H₂O₂ generation in endothelial cells.     

Ca2+ channels at the plasma membrane of stomatal guard cells are activated by hyperpolarization and abscisic acid

In stomatal guard cells of higher-plant leaves, abscisic acid (ABA) evokes increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) by means of Ca(2+) entry from outside and release from intracellular stores. The mechanism(s) for Ca(2+) flux across the plasma membrane is poorly understood. Because [Ca(2+)](i) increases are voltage-sensitive, we suspected a Ca(2+) channel at the guard cell plasma membrane that activates on hyperpolarization and is regulated by ABA. We recorded single-channel currents across the Vicia guard cell plasma membrane using Ba(2+) as a charge-carrying ion. Both cell-attached and excised-patch measurements uncovered single-channel events with a maximum conductance of 12.8 +/- 0.4 pS and a high selectivity for Ba(2+) (and Ca(2+)) over K(+) and Cl(-). Unlike other Ca(2+) channels characterized to date, these channels rectified strongly toward negative voltages with an open probability (P(o)) that increased with [Ba(2+)] outside and decreased roughly 10-fold when [Ca(2+)](i) was raised from 200 nM to 2 microM. Adding 20 microM ABA increased P(o), initially by 63- to 260-fold; in both cell-attached and excised patches, it shifted the voltage sensitivity for channel activation, and evoked damped oscillations in P(o) with periods near 50 s. A similar, but delayed response was observed in 0.1 microM ABA. These results identify a Ca(2+)-selective channel that can account for Ca(2+) influx and increases in [Ca(2+)](i) triggered by voltage and ABA, and they imply a close physical coupling at the plasma membrane between ABA perception and Ca(2+) channel control.     

Arabidopsis group C Raf-like protein kinases negatively regulate abscisic acid signaling and are direct substrates of SnRK2

The phytohormone abscisic acid (ABA) plays a major role in abiotic stress responses in plants, and subclass III SNF1-related protein kinase 2 (SnRK2) kinases mediate ABA signaling. In this study, we identified Raf36, a group C Raf-like protein kinase in Arabidopsis, as a protein that interacts with multiple SnRK2s. A series of reverse genetic and biochemical analyses revealed that 1) Raf36 negatively regulates ABA responses during postgermination growth, 2) the N terminus of Raf36 is directly phosphorylated by SnRK2s, and 3) Raf36 degradation is enhanced in response to ABA. In addition, Raf22, another C-type Raf-like kinase, functions partially redundantly with Raf36 to regulate ABA responses. A comparative phosphoproteomic analysis of ABA-induced responses of wild-type and raf22raf36-1 plants identified proteins that are phosphorylated downstream of Raf36 and Raf22 in planta. Together, these results support a model in which Raf36/Raf22 function mainly under optimal conditions to suppress ABA responses, whereas in response to ABA, the SnRK2 module promotes Raf36 degradation as a means of alleviating Raf36-dependent inhibition and allowing for heightened ABA signaling to occur.

Environmental stresses, such as drought, high salinity, and low temperature, have adverse effects on plant growth and development. Abscisic acid (ABA) is a phytohormone that plays important roles in responses and adaptations to these stresses, as well as in embryo maturation and seed dormancy (1, 2). The major ABA signaling pathway consists of three core components: ABA receptors, type 2C protein phosphatases (PP2Cs), and SNF1-related protein kinase 2s (SnRK2s) (3, 4). In this pathway, SnRK2s transmit ABA- or osmostress-induced signals through phosphorylation of downstream substrates, thereby promoting ABA- or stress-inducible gene expression and stomatal closure (5–8). The Arabidopsis genome contains 10 members of SnRK2, and they are classified into three subclasses (9, 10). Among them, subclass III members, SRK2D/SnRK2.2, SRK2E/OST1/SnRK2.6, and SRK2I/SnRK2.3, are essential for ABA responses (11–14).Raf-like protein kinases were recently identified as regulators of ABA signaling. Among 80 putative mitogen-activated protein kinase kinase kinases (MAPKKKs) in Arabidopsis, 48 members are categorized as Raf-like subfamilies and can further be divided into 11 subgroups (B1 to B4 and C1 to C7) (15). In Physcomitrella patens, the ABA and abiotic stress-responsive Raf-like kinase (ARK) gene, also named as ABA nonresponsive (ANR) or constitutive triple response 1-like (CTR1L), is required for ABA-responsive SnRK2-activation, gene expression and drought, osmotic and freezing tolerance (16–18). ARK encodes a B3 subgroup Raf-like protein kinase that phosphorylates SnRK2s in vitro, suggesting that ARK functions as an upstream kinase of SnRK2s (16). In addition, several recent studies also confirmed that Arabidopsis B2, B3, and B4 subgroup of Raf-like kinases are required for osmotic stress–induced SnRK2 activation, gene expression, and stomatal closure (19–23). In addition to group B, it had been reported that several group C Raf-like kinases are associated with ABA responses. For example, Arabidopsis Raf43, a C5 kinase, regulates ABA sensitivity during seed germination and seedling root growth (24), whereas Raf22, a member of C6 subgroup, negatively regulates stress- or ABA-induced growth arrest (25). However, despite these ABA-related genetic phenotypes, it is still unclear whether group C kinases directly regulate SnRK2-dependent signaling pathways.In this study, we identified Raf36, a C5 group Raf-like kinase, as a protein that directly interacts with, and is phosphorylated by, SnRK2. Our evidence indicates that Raf36 functions as a negative regulator of ABA signaling pathway during the postgerminative growth stage under the control of SnRK2. In addition, we revealed that Raf22, a C6 Raf-like kinase, functions partially redundantly with Raf36. Raf36 mainly functions under optimal conditions, because its degradation was promoted under ABA treatment. Furthermore, comparative phosphoproteomic analysis unveiled the phosphorylation network regulated by Raf22 and/or Raf36 in planta. Collectively, unlike group B Rafs, which have been recently reported as an “accelerator” of ABA response upstream of SnRK2s, our results demonstrate that Arabidopsis group C Rafs, Raf22 and Raf36, function as a “brake” of ABA response downstream of SnRK2s.     

Role of nitric oxide signaling components in differentiation of embryonic stem cells into myocardial cells

Nitric oxide (NO) is involved in number of physiological and pathological events. Our previous studies demonstrated a differential expression of NO signaling components in mouse and human ES cells. Here, we demonstrate the effect of NO donors and soluble guanylyl cyclase (sGC) activators in differentiation of ES cells into myocardial cells. Our results with mouse and human ES cells demonstrate an increase in Nkx2.5 and myosin light chain (MLC2) mRNA expression on exposure of cells to NO donors and a decrease in mRNA expression of both cardiac-specific genes with nonspecific NOS inhibitor and a concomitant increase and decrease in the mRNA levels of sGC α₁ subunit. Although sGC activators alone exhibited an increase in mRNA expression of cardiac genes (MLC2 and Nkx2.5), robust inductions of mRNA and protein expression of marker genes were observed when NO donors and sGC activators were combined. Measurement of NO metabolites revealed an increase in the nitrite levels in the conditioned media and cell lysates on exposure of cells to the different concentrations of NO donors. cGMP analysis in undifferentiated stem cells revealed a lack of stimulation with NO donors. Differentiated cells however, acquired the ability to be stimulated by NO donors. Although, 3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo [3,4-b]pyridine (BAY 41-2272) alone was able to stimulate cGMP accumulation, the combination of NO donors and BAY 41-2272 stimulated cGMP levels more than either of the agents separately. These studies demonstrate that cGMP-mediated NO signaling plays an important role in the differentiation of ES cells into myocardial cells.     

Water-stress-induced changes in the abscisic acid content of guard cells and other cells of Vicia faba L. leaves as determined by enzyme-amplified immunoassay

A highly sensitive, solid-phase, enzyme-amplified immunoassay for the plant growth regulator (+)-abscisic acid (ABA) was developed. The assay sensitivity (0.2-10 fmol) was sufficient for analyzing free ABA in homogeneous tissue samples dissected from Vicia faba L. leaves. Eight hours after detached leaves had been desiccated to 10% loss of fresh weight, the bulk leaf ABA content increased from ≤0.2 to 6.2 ng·(mg dry weight)^-1. Epidermal tissue, spongy parenchyma cells, and palisade parenchyma cells from this water-stressed leaf had the following ABA contents, respectively: 4.8, 9.4, and 9.0 ng·(mg dry weight)^-1. Guard cells, which respond to exogenous ABA by losing solutes and volume, were also assayed. When they were dissected from control (fully hydrated) leaves, their ABA content was ≈0.7 fg·(cell pair)^-1 [[unk]0.2 ng·(mg dry weight)^-1]. In contrast, the ABA content of guard cells of water-stressed leaves was ≈17.7 fg·(cell pair)^-1. These results indicate that ABA accumulation in a highly stressed V. faba leaflet is generalized; guard cells contain only 0.15% of bulk leaf ABA. The time course for loss of ABA from guard cells of a floating epidermal peel was studied. There was little loss within 30 min, but after 4 hr, the ABA content was only 17% of the original value. These results indicate that the bulk of guard cell ABA is not readily diffusible (i.e., probably not apoplastic). The results also indicate that common laboratory procedures results in lowered guard cell ABA content.