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Environmental stimuli-triggered stomatal movement is a key physiological process that regulates CO 2 uptake and water loss in plants. Stomata are defined by pairs of guard cells that perceive and transduce external signals, leading to cellular volume changes and consequent stomatal aperture change. Within the visible light spectrum, red light induces stomatal opening in intact leaves. However, there has been debate regarding the extent to which red-light-induced stomatal opening arises from direct guard cell sensing of red light versus indirect responses as a result of red light influences on mesophyll photosynthesis. Here we identify conditions that result in red-light-stimulated stomatal opening in isolated epidermal peels and enlargement of protoplasts, firmly establishing a direct guard cell response to red light. We then employ metabolomics workflows utilizing gas chromatography mass spectrometry and liquid chromatography mass spectrometry for metabolite profiling and identification of Arabidopsis guard cell metabolic signatures in response to red light in the absence of the mesophyll. We quantified 223 metabolites in Arabidopsis guard cells, with 104 found to be red light responsive. These red-light-modulated metabolites participate in the tricarboxylic acid cycle, carbon balance, phytohormone biosynthesis and redox homeostasis. We next analyzed selected Arabidopsis mutants, and discovered that stomatal opening response to red light is correlated with a decrease in guard cell abscisic acid content and an increase in jasmonic acid content. The red-light-modulated guard cell metabolome reported here provides fundamental information concerning autonomous red light signaling pathways in guard cells., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Effects of exogenous abscisic acid (ABA) and CaCl2 on γ-aminobutyric acid (GABA) accumulation of germinated fava bean under hypoxia-NaCl stress were investigated. Exogenous ABA resulted in the enhancement of glutamate decarboxylase (GAD) and diamine oxidase (DAO) activity as well as GABA content in cotyledon and shoot. CaCl2 increased both enzyme activities in shoot and GABA content in cotyledon and shoot. ABA downregulated GAD expression in cotyledon and radicle, while upregulated that in shoot; it also upregulated DAO expression in each organ. CaCl2 upregulated GAD expression in cotyledon, while downregulated that in radicle. However, it upregulated DAO expression in shoot, downregulated that in radicle. ABA inhibitor fluridon and ethylenediaminetetraacetic acid inhibited GAD and DAO activities significantly so that inhibited GABA accumulation through reducing ABA biosynthesis and chelating Ca(2+), respectively. However, they upregulated GAD and DAO expression in varying degrees. These results indicate that ABA and Ca(2+) participate in GABA biosynthesis in fava bean during germination under hypoxia-NaCl stress.

Plants developed under constant high (> 85%) relative air humidity (RH) have larger stomata that are unable to close completely. One of the hypotheses for the less responsive stomata is that the plants have reduced sensitivity to abscisic acid (ABA). Both ABA and darkness are signals for stomatal closure and induce the production of the secondary messenger hydrogen peroxide (H2O2). In this study, the ability of Vicia faba plants developed in moderate or high RH to close the stomata in response to darkness, ABA and H2O2 was investigated. Moreover, the ability of the plants to produce H2O2 when treated with ABA or transferred to darkness was also assessed. Our results show that the ABA concentration in moderate RH is not increased during darkness even though the stomata are closing. This indicates that stomatal closure in V. faba during darkness is independent of ABA production. ABA induced both H2O2 production and stomatal closure in stomata formed at moderate RH. H2O2 production, as a result of treatment with ABA, was also observed in stomata formed at high RH, though the closing response was considerably smaller as compared with moderate RH. In either RH, leaf ABA concentration was not affected by darkness. Similarly to ABA treatment, darkness elicited both H2O2 production and stomatal closure following plant cultivation at moderate RH. Contrary to this, neither H2O2 production nor stomatal closure took place when stomata were formed at high RH. These results suggest that the reduced stomatal response in plants developed in continuous high RH is caused by one or more factors downstream of H2O2 in the signaling pathway toward stomatal closure.

An abscisic acid (ABA)-insensitive Vicia faba mutant, fia (fava bean impaired in ABA-induced stomatal closure) had previously been isolated. In this study, it was investigated how FIA functions in ABA signalling in guard cells of Vicia faba. Unlike ABA, methyl jasmonate (MeJA), H(2)O(2), and nitric oxide (NO) induced stomatal closure in the fia mutant. ABA did not induce production of either reactive oxygen species or NO in the mutant. Moreover, ABA did not suppress inward-rectifying K(+) (K(in)) currents or activate ABA-activated protein kinase (AAPK) in mutant guard cells. These results suggest that FIA functions as an early signal component upstream of AAPK activation in ABA signalling but does not function in MeJA signalling in guard cells of Vicia faba.

Abscisic acid (ABA) signaling is important for stress responses and developmental processes in plants. A subgroup of protein phosphatase 2C (group A PP2C) or SNF1-related protein kinase 2 (subclass III SnRK2) have been known as major negative or positive regulators of ABA signaling, respectively. Here, we demonstrate the physical and functional linkage between these two major signaling factors. Group A PP2Cs interacted physically with SnRK2s in various combinations, and efficiently inactivated ABA-activated SnRK2s via dephosphorylation of multiple Ser/Thr residues in the activation loop. This step was suppressed by the RCAR/PYR ABA receptors in response to ABA. However the abi1-1 mutated PP2C did not respond to the receptors and constitutively inactivated SnRK2. Our results demonstrate that group A PP2Cs act as 'gatekeepers' of subclass III SnRK2s, unraveling an important regulatory mechanism of ABA signaling.

SB203580 is a specific inhibitor of p38 mitogen-activated protein (MAP) kinase and has been widely used to investigate the physiological roles of p38 in animal and yeast cells. Here by using an epidermal strip bioassay, laser-scanning confocal microscopy and whole-cell patch clamp analysis, we assess the effects of pyridinyl imidazoles-like SB203580 on the H(2)O(2) signaling in guard cells of Vicia faba L. The results indicated that SB203580 blocks H(2)O(2)- or ABA-induced stomatal closure, ABA-induced H(2)O(2) generation, and decrease in K(+) fluxing across plasma membrane of Vicia guard cells by application of ABA and H(2)O(2), whereas its analog SB202474 had no effect on these events. Thus, these results suggest that activation of p38-like MAP kinase modulates guard cell ROS signaling in response to stress.

The Arabidopsis thaliana RNA binding protein UBA2a is the closest homologue of the Vicia faba AKIP1 (56% identity). Like AKIP1, UBA2a is a constitutively-expressed nuclear protein and in response to ABA it is also reorganized within the nucleus in "speckles" suggesting a possible role of this protein in the regulation of mRNA metabolism during ABA signaling. AKIP1 interacts with, and is phosphorylated by, the upstream ABA-activated protein kinase AAPK. We have investigated if a pathway similar to that described in Vicia faba also exists in Arabidopsis. Our results showed that despite the resemblance between the corresponding Vicia and Arabidopsis proteins, it appears that the function of UBA2a is independent of OST1 phosphorylation.

The phytohormone abscisic acid (ABA) reports on the water status of the plant and induces stomatal closure. Guard cell anion channels play a central role in this response, because they mediate anion efflux, and in turn, cause a depolarization-induced K+ release. We recorded early steps in ABA signaling, introducing multibarreled microelectrodes in guard cells of intact plants. Upon external ABA treatment, anion channels transiently activated after a lag phase of approximately 2 min. As expected for a cytosolic ABA receptor, iontophoretic ABA loading into the cytoplasm initiated a rise in anion current without delay. These ABA responses could be elicited repetitively at resting and at largely depolarized potentials (e.g., 0 mV), ruling out signal transduction by means of hyperpolarization-activated calcium channels. Likewise, ABA stimulation did not induce a rise in the cytosolic free-calcium concentration. However, the presence of approximately 100 nM background Ca2+ was required for anion channel function, because the action of ABA on anion channels was repressed after loading of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate. The chain of events appears very direct, because none of the tested putative ABA-signaling intermediates (inositol 1,4,5 trisphosphate, inositol hexakisphosphate, nicotinic acid adenine dinucleotide phosphate, and cyclic ADP-ribose), could mimic ABA as anion channel activator. In patch-clamp experiments, cytosolic ABA also evoked anion current transients carried by R- and S-type anion channels. The response was dose-dependent with half-maximum activation at 2.6 microM ABA. Our studies point to an ABA pathway initiated by ABA binding to a cytosolic receptor that within seconds activates anion channels, and in turn, leads to depolarization of the plasma membrane.

It is usually suggested that either H(2)O(2) or NO function as a signal molecule in mediating the ABA-induced stomatal closure of guard cells, but there has been no report on the relationship between H(2)O(2) and NO in ABA signal transduction pathway. Here, using stomatal analysis and laser scanning cofocal microscope techniques, we show firstly that NO functions as a downstream intermediate of H(2)O(2) signaling to mediate ABA-induced stomatal closure in Vicia faba L. Sodium nitroprusside (SNP, a NO donor) and H(2)O(2) can mimic the effects of ABA on stomatal closure. Carboxy-PTIO (c-PTIO, a specific scavenger of NO) partly reverse the stomatal closure induced by ABA or H(2)O(2), while catalase (CAT), a H(2)O(2) scavenger, failed to reverse the NO-induced aperture reduction in Vicia faba guard cells. Monitoring the changes in both NO and H(2)O(2) generation in guard cells by using fluorescent probe of NO or H(2)O(2), DAF-2DA or H2DCFDA, respectively, we found that the generating rate of H(2)O(2) in guard cells was faster than that of NO after being treated with ABA 10 micromol/L. CAT almost completely inhibited the increase in DAF fluorescence induced by ABA. Similar to ABA, exogenous H(2)O(2) provoked the production of NO. c-PTIO slightly enhanced the fluorescent intensity of DCF stimulated by ABA, while exogenous SNP did not increase DCF fluorescence in guard cells. Taken together, these results suggest that H(2)O(2) could probably act as upstream component of NO signaling and NO negatively regulate H(2)O(2) generation during ABA-induced stomatal closure in guard cells.

Blue light (BL)-dependent H+ pumping by guard cells, which drives stomatal opening, is inhibited by abscisic acid (ABA). We investigated this response with respect to the activity of plasma membrane H+-ATPase using Vicia guard cell protoplasts. ATP hydrolysis by the plasma membrane H+-ATPase, phosphorylation of the H+-ATPase, and the binding of 14-3-3 protein to the H+-ATPase stimulated by BL were inhibited by ABA at 10 microm. All of these responses were similarly inhibited by hydrogen peroxide (H2O2) at 1 mm. The ABA-induced inhibitions of BL-dependent H+ pumping and phosphorylation of the H+-ATPase were partially restored by ascorbate, an intracellular H2O2 scavenger. A single-cell analysis of the cytosolic H2O2 using 2',7'-dichlorofluorescin revealed that H2O2 was generated by ABA in guard cell protoplasts. We also indicated that H+ pumping induced by fusicoccin and the binding of 14-3-3 protein to the H+-ATPase were inhibited slightly (approximately 20%) by both ABA and H2O2. By contrast, H2O2 at 1 mm did not affect H+ pumping by the H+-ATPase in microsomal membranes. From these results, we concluded that inhibition of BL-dependent H+ pumping by ABA was due to a decrease in the phosphorylation levels of H+-ATPase and that H2O2 might be involved in this response. Moreover, there are at least two inhibition sites by ABA in the BL signaling pathway of guard cells.

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.

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.

We isolated a mutant from Vicia faba L. cv. House Ryousai. It wilts easily under strong light and high temperature conditions, suggesting that its stomatal movement may be disturbed. We determined responses of mutant guard cells to some environmental stimuli. Mutant guard cells demonstrated an impaired ability to respond to ABA in 0.1 mM CaCl(2) and stomata did not close in the presence of up to 1 mM ABA, whereas wild-type stomata closed when exposed to 10 micro M ABA. Elevating external Ca(2+) caused a similar degree of stomatal closure in the wild type and the mutant. A high concentration of CO(2) (700 micro l liter(-1)) induced stomatal closure in the wild type, but not in the mutant. On the basis of these results, we propose the working hypothesis that the mutation occurs in the region downstream of CO(2) and ABA sensing and in the region upstream of Ca(2+) elevation. The mutant is named fia (fava bean impaired in ABA-induced stomatal closure).

Abscisic acid (ABA) is a phytohormone that plays a key role as a stress signal, regulating water relations during drought conditions, by inducing stomatal closure. However, to date, no putative ABA receptor(s) has been reported at the protein sequence, gene family, or cellular localization levels. We used biotinylated ABA (bioABA) to characterize the ABA-perception sites in the stomatal guard cells of Vicia faba. Treatment with bioABA induced stomatal closure and shrinkage of guard cell protoplasts (GCPs). The ABA-perception sites were visualized by fluorescence microscopy and confocal laser scanning microscopy (CLSM), using bioABA and fluorescence-labeled avidin. Fluorescent particles were observed in patches on the surface of the GCPs. Fluorescence intensity was quantified by flow cytometry (FCM) as well as by CLSM. Binding of bioABA was inhibited by ABA in a dose-dependent manner. Pre-treatment of GCPs with proteinase K also blocked the binding of bioABA. Binding of bioABA was inhibited by RCA-7a, an ABA analog that induces stomatal closure, but not by RCA-16, which has no effect on stomatal aperture. Another ABA analog, PBI-51, inhibited ABA-induced stomatal closure. This ABA antagonist also inhibited binding of bioABA to the GCPs. These results suggest that ABA is perceived on the plasma membrane of stomatal guard cells, and that the present experimental methods constitute valuable tools for characterizing the nature of the ABA receptor(s) that perceives physiological ABA signals. These imaging studies allow us to demonstrate the spatial distribution of the ABA-perception sites. Visualization of the ABA-perception sites provides new insights into the nature of membrane-associated ABA receptor(s).

The methods of confocal laser scanning microscopy (CLSM) and microinjection were used to study ABA-induced H2O2 in guard cells (Vicia faba), which were labeled with H2O2 specific probe-2, 7-dichlorofluorescin diacetate(H2DCFDA). The results indicated 100 U/mL catalase (CAT) could inhibit partly stomatal closure induced by ABA. 10(-3) mmol/L ABA could significantly induce H2O2 production in chloroplast in guard cells of Vicia faba following microinjection, and 100 U/mL CAT could partly abolish the effects following simultaneous microinjection of ABA and CAT. These suggest that H2O2 is possibly involved in ABA signaling leading to stomatal closure.