Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba

One of the most important functions of the plant hormone abscisic acid (ABA) is to induce stomatal closure by reducing the turgor of guard cells under water deficit. Under environmental stresses, hydrogen peroxide (H(2)O(2)), an active oxygen species, is widely generated in many biological systems. Here, using an epidermal strip bioassay and laser-scanning confocal microscopy, we provide evidence that H(2)O(2) may function as an intermediate in ABA signaling in Vicia faba guard cells. H(2)O(2) inhibited induced closure of stomata, and this effect was reversed by ascorbic acid at concentrations lower than 10(-5) M. Further, ABA-induced stomatal closure also was abolished partly by addition of exogenous catalase (CAT) and diphenylene iodonium (DPI), which are an H(2)O(2) scavenger and an NADPH oxidase inhibitor, respectively. Time course experiments of single-cell assays based on the fluorescent probe dichlorofluorescein showed that the generation of H(2)O(2) was dependent on ABA concentration and an increase in the fluorescence intensity of the chloroplast occurred significantly earlier than within the other regions of guard cells. The ABA-induced change in fluorescence intensity in guard cells was abolished by the application of CAT and DPI. In addition, ABA microinjected into guard cells markedly induced H(2)O(2) production, which preceded stomatal closure. These effects were abolished by CAT or DPI micro-injection. Our results suggest that guard cells treated with ABA may close the stomata via a pathway with H(2)O(2) production involved, and H(2)O(2) may be an intermediate in ABA signaling.     

Carbon monoxide-induced stomatal closure in Vicia faba is dependent on nitric oxide synthesis

Recently, in animals, carbon monoxide (CO), like nitric oxide (NO), was implicated as another important physiological messenger or bioactive molecule. Previous researches indicate that heme oxygenase (HO)-1 (EC 1.14.99.3) catalyzes the oxidative conversion of heme to CO and biliverdin IXa (BV) with the concomitant release of iron. However, little is known about the physiological roles of CO in plant, especially in stomatal movement of guard cells. In the present paper, the regulatory role of CO during stomatal movement in Vicia faba was surveyed. Results indicated that, like sodium nitroprusside (SNP), CO donor hematin induced stomatal closure in dose- and time-dependent manners. These responses were also proved by the addition of gaseous CO aqueous solution with different concentrations, showing for the first time that CO and NO exhibit similar regulation role in the stomatal movement. Moreover, our data showed that 2,4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO)/N^G-nitro- l -arginine-methyl ester ( l -NAME) not only reversed stomatal closure by CO, but also suppressed the NO fluorescence induced by CO, implying that CO-induced stomatal closure probably involves NO/nitric oxide synthase (NOS) signal system. Additionally, the CO/NO scavenger hemoglobin (Hb) and CO-specific synthetic inhibitor zinc protoporphyrin IX (ZnPPIX), NO scavenger cPTIO and NOS inhibitor l -NAME reversed the darkness-induced stomatal closure and NO fluorescence. These results show that, maybe like NO, the levels of CO in guard cells of V.   faba is higher in dark than that in light, HO-1 and NOS are the enzyme systems responsible for generating endogenous CO and NO in darkness, respectively, and that CO being from HO-1 mediates darkness-induced NO synthesis in guard cells' stomatal closure of V.   faba .     

Glucose‐ and mannose‐induced stomatal closure is mediated by ROS production,Ca2+ and water channel in Vicia faba

Sugars act as vital signaling molecules that regulate plant growth, development and stress responses. However, the effects of sugars on stomatal movement have been unclear. In our study, we explored the effects of monosaccharides such as glucose and mannose on stomatal aperture. Here, we demonstrate that glucose and mannose trigger stomatal closure in a dose‐ and time‐dependent manner in epidermal peels of broad bean (Vicia faba). Pharmacological studies revealed that glucose‐ and mannose‐induced stomatal closure was almost completely inhibited by two reactive oxygen species (ROS) scavengers, catalase (CAT) and reduced glutathione (GSH), was significantly abolished by an NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI), whereas they were hardly affected by a peroxidase inhibitor, salicylhydroxamic acid (SHAM). Furthermore, glucose‐ and mannose‐induced stomatal closure was strongly inhibited by a Ca²⁺ channel blocker, LaCl₃, a Ca²⁺ chelator, ethyleneglycol‐bis(beta‐aminoethylether)‐N,N'‐tetraacetic acid (EGTA) and two water channel blockers, HgCl₂ and dimethyl sulfoxide (DMSO); whereas the inhibitory effects of the water channel blockers were essentially abolished by the reversing agent β‐mercaptoethanol (β‐ME). These results suggest that ROS production mainly via NADPH oxidases, Ca²⁺ and water channels are involved in glucose‐ and mannose‐induced stomatal closure.     

Nitric oxide, stomatal closure, and abiotic stress

Various data indicate that nitric oxide (NO) is an endogenoussignal in plants that mediates responses to several stimuli.Experimental evidence in support of such signalling roles forNO has been obtained via the application of NO, usually in theform of NO donors, via the measurement of endogenous NO, andthrough the manipulation of endogenous NO content by chemicaland genetic means. Stomatal closure, initiated by abscisic acid(ABA), is effected through a complex symphony of intracellularsignalling in which NO appears to be one component. ExogenousNO induces stomatal closure, ABA triggers NO generation, removalof NO by scavengers inhibits stomatal closure in response toABA, and ABA-induced stomatal closure is reduced in mutantsthat are impaired in NO generation. The data indicate that ABA-inducedguard cell NO generation requires both nitric oxide synthase-likeactivity and, in Arabidopsis, the NIA1 isoform of nitrate reductase(NR). NO stimulates mitogen-activated protein kinase (MAPK)activity and cGMP production. Both these NO-stimulated eventsare required for ABA-induced stomatal closure. ABA also stimulatesthe generation of H₂O₂ in guard cells, and pharmacological andgenetic data demonstrate that NO accumulation in these cellsis dependent on such production. Recent data have extended thismodel to maize mesophyll cells where the induction of antioxidantdefences by water stress and ABA required the generation ofH₂O₂ and NO and the activation of a MAPK. Published data suggestthat drought and salinity induce NO generation which activatescellular processes that afford some protection against the oxidativestress associated with these conditions. Exogenous NO can alsoprotect cells against oxidative stress. Thus, the data suggestan emerging model of stress responses in which ABA has severalameliorative functions. These include the rapid induction ofstomatal closure to reduce transpirational water loss and theactivation of antioxidant defences to combat oxidative stress.These are two processes that both involve NO as a key signallingintermediate. Key words: Abscisic acid, antioxidants, guard cells, hydrogen peroxide, nitric oxide, oxidative stress, stomata, water stress Received 19 June 2007; Revised 21 September 2007 Accepted 5 November 2007     

Exogenous application of trehalose induced H2O2 production and stomatal closure in Vicia faba

Trehalose can reduce stomatal aperture by a hydrogen-peroxide-dependent pathway in Vicia faba L. (cv. Daqingpi) resulting in significantly lower values of net photosynthetic rate (P_N), stomatal conductance (g_s), and transpiration rate (E). At 8 and 24 h, the lower P_N in trehalose-treated plants was accompanied by significant decrease in intercellular CO₂ concentration (c_i) suggesting that the reduction of P_N was caused by stomatal limitation. At 48 and 72 h, trehalose decreased apparent carboxylation efficiency (P_N/c_i) and did not decrease c_i and g_s compared with controls; therefore the reduction in photosynthesis was caused by non-stomatal limitation. Trehalose treatment resulted in significantly higher effective photochemical efficiency of PS II (Φ_PSII) and did not affect maximum photochemical efficiency of PS II (F_v/F_m). At 24, 48, and 72 h, trehalose decreased non-photochemical quenching (NPQ) and increased photochemical quenching (qP). Our results suggest that trehalose did not damage photosynthetic reaction centers.     

Hydrogen peroxide generated by copper amine oxidase is involved in abscisic acid-induced stomatal closure in Vicia faba

H₂O₂ is an essential signal in absicic acid (ABA)-induced stomatalclosure. It can be synthesized by several enzymes in plants.In this study, the roles of copper amine oxidase (CuAO) in H₂O₂production and stomatal closure were investigated. ExogenousABA stimulated apoplast CuAO activity, increased H₂O₂ productionand [Ca²⁺]_cyt levels in Vicia faba guard cells, and inducedstomatal closure. These processes were impaired by CuAO inhibitor(s).In the metabolized products of CuAO, only H₂O₂ could inducestomatal closure. By the analysis of enzyme kinetics and polyaminecontents in leaves, putrescine was regarded as a substrate ofCuAO. Putrescine showed similar effects with ABA on the regulationof H₂O₂ production, [Ca²⁺]_cyt levels, as well as stomatal closure.The results suggest that CuAO in V. faba guard cells is an essentialenzymatic source for H₂O₂ production in ABA-induced stomatalclosure via the degradation of putrescine. Calcium messengeris an important intermediate in this process. Key words: Abscisic acid, calcium, copper amine oxidase, hydrogen peroxide, putrescine, stomatal closure, Vicia faba Received 13 October 2007; Revised 16 December 2007 Accepted 20 December 2007     

Inhibition of dark‐induced stomatal closure by fusicoccin involves a removal of hydrogen peroxide in guard cells of Vicia faba

Fusicoccin (FC) treatment prevents dark‐induced stomatal closure, the mechanism of which is still obscure. By using pharmacological approaches and laser‐scanning confocal microscopy, the relationship between FC inhibition of dark‐induced stomatal closure and the hydrogen peroxide (H₂O₂) levels in guard cells in broad bean was studied. Like ascorbic acid (ASA), a scavenger of H₂O₂ and diphenylene iodonium (DPI), an inhibitor of H₂O₂‐generating enzyme NADPH oxidase, FC was found to inhibit stomatal closure and reduce H₂O₂ levels in guard cells in darkness, indicating that FC‐caused inhibition of dark‐induced stomatal closure is related to the reduction of H₂O₂ levels in guard cells. Furthermore, like ASA, FC not only suppressed H₂O₂‐induced stomatal closure and H₂O₂ levels in guard cells treated with H₂O₂ in light, but also reopened the stomata which had been closed by darkness and reduced the level of H₂O₂ that had been generated by darkness, showing that FC causes H₂O₂ removal in guard cells. The butyric acid treatment simulated the effects of FC on the stomata treated with H₂O₂ and had been closed by dark, and on H₂O₂ levels in guard cells of stomata treated with H₂O₂ and had been closed by dark, and both FC and butyric acid reduced cytosol pH in guard cells of stomata treated with H₂O₂ and had been closed by dark, which demonstrates that cytosolic acidification mediates FC‐induced H₂O₂ removal. Taken together, our results provide evidence that FC causes cytosolic acidification, consequently induces H₂O₂ removal, and finally prevents dark‐induced stomatal closure.     

Nitric oxide involved in the IL‐1β‐induced inhibition of fructose intestinal transport

Interleukin‐1β (IL‐1β) is a pleiotropic cytokine produced by cells of the immune system and a large variety of other cell types including endothelial cells. It is released during inflammatory and infectious diseases, and possesses a wide spectrum of autocrine, paracrine and endocrine activities. The aim of this work was to examine the IL‐1β effect on D ‐fructose transport across rabbit jejunum and try to identify the mediators implicated in this process. A sepsis condition was induced for 90 min after intravenous (iv) administration of IL‐1β and body temperature was recorded. Studies on cellular intestinal integrity have not shown modifications of the epithelium and the basement membrane. D ‐fructose intestinal transport was studied in rabbit jejunum from control and treated animals and it was reduced in the latter ones. This cytokine decreased both the mucosal to serosal transepithelial flux and the transport across brush‐border membrane vesicles of D ‐fructose. The inhibition was reversed by L ‐NAME (nitric oxide [NO] synthase inhibitor), but not by indomethacin (cyclooxygenase 1 and 2 inhibitor). Both inhibitors were administered iv 15 min before the IL‐1β. The protein levels of GLUT5 were not changed in all animal groups and those of mRNA were even increased. In summary, these findings indicate that IL‐1β, at the time assayed, induced a significant reduction in the relative intrinsic activity of GLUT5 and in this decrease are involved NO signalling pathways. In this way, blockage of D ‐fructose intestinal uptake by IL‐1β may be playing an essential role in the pathophysiology of septic shock. J. Cell. Biochem. 111: 1321–1329, 2010. © 2010 Wiley‐Liss, Inc.     

ARP2/3 complex‐mediated actin dynamics is required for hydrogen peroxide‐induced stomatal closure in Arabidopsis

Multiple cellular events like dynamic actin reorganization and hydrogen peroxide (H₂O₂) production were demonstrated to be involved in abscisic acid (ABA)‐induced stomatal closure. However, the relationship between them as well as the underlying mechanisms remains poorly understood. Here, we showed that H₂O₂ generation is indispensable for ABA induction of actin reorganization in guard cells of Arabidopsis that requires the presence of ARP2/3 complex. H₂O₂‐induced stomatal closure was delayed in the mutants of arpc4 and arpc5, and the rate of actin reorganization was slowed down in arpc4 and arpc5 in response to H₂O₂, suggesting that ARP2/3‐mediated actin nucleation is required for H₂O₂‐induced actin cytoskeleton remodelling. Furthermore, the expression of H₂O₂ biosynthetic related gene AtrbohD and the accumulation of H₂O₂ was delayed in response to ABA in arpc4 and arpc5, demonstrating that misregulated actin dynamics affects H₂O₂ production upon ABA treatment. These results support a possible causal relation between the production of H₂O₂ and actin dynamics in ABA‐mediated guard cell signalling: ABA triggers H₂O₂ generation that causes the reorganization of the actin cytoskeleton partially mediated by ARP2/3 complex, and ARP2/3 complex‐mediated actin dynamics may feedback regulate H₂O₂ production.     

Osmo-sensitive and stretch-activated calcium-permeable channels in Vicia faba guard cells are regulated by actin dynamics

In responses to a number of environmental stimuli, changes of cytoplasmic [Ca(2+)](cyt) in stomatal guard cells play important roles in regulation of stomatal movements. In this study, the osmo-sensitive and stretch-activated (SA) Ca(2+) channels in the plasma membrane of Vicia faba guard cells are identified, and their regulation by osmotic changes and actin dynamics are characterized. The identified Ca(2+) channels were activated under hypotonic conditions at both whole-cell and single-channel levels. The channels were also activated by a stretch force directly applied to the membrane patches. The channel-mediated inward currents observed under hypotonic conditions or in the presence of a stretch force were blocked by the Ca(2+) channel inhibitor Gd(3+). Disruption of actin filaments activated SA Ca(2+) channels, whereas stabilization of actin filaments blocked the channel activation induced by stretch or hypotonic treatment, indicating that actin dynamics may mediate the stretch activation of these channels. In addition, [Ca(2+)](cyt) imaging demonstrated that both the hypotonic treatment and disruption of actin filaments induced significant Ca(2+) elevation in guard cell protoplasts, which is consistent with our electrophysiological results. It is concluded that stomatal guard cells may utilize SA Ca(2+) channels as osmo sensors, by which swelling of guard cells causes elevation of [Ca(2+)](cyt) and consequently inhibits overswelling of guard cells. This SA Ca(2+) channel-mediated negative feedback mechanism may coordinate with previously hypothesized positive feedback mechanisms and regulate stomatal movement in response to environmental changes.     

WAVE, a novel WASP-family protein involved in actin reorganization induced by Rac.

Rac is a Rho-family small GTPase that induces the formation of membrane ruffles. However, it is poorly understood how Rac-induced reorganization of the actin cytoskeleton, which is essential for ruffle formation, is regulated. Here we identify a novel Wiskott-Aldrich syndrome protein (WASP)-family protein, WASP family Verprolin-homologous protein (WAVE), as a regulator of actin reorganization downstream of Rac. Ectopically expressed WAVE induces the formation of actin filament clusters that overlap with the expressed WAVE itself. In this actin clustering, profilin, a monomeric actin-binding protein that has been suggested to be involved in actin polymerization, was shown to be essential. The expression of a dominant-active Rac mutant induces the translocation of endogenous WAVE from the cytosol to membrane ruffling areas. Furthermore, the co-expression of a deltaVPH WAVE mutant that cannot induce actin reorganization specifically suppresses the ruffle formation induced by Rac, but has no effect on Cdc42-induced actin-microspike formation, a phenomenon that is also known to be dependent on rapid actin reorganization. The deltaVPH WAVE also suppresses membrane-ruffling formation induced by platelet-derived growth factor in Swiss 3T3 cells. Taken together, we conclude that WAVE plays a critical role downstream of Rac in regulating the actin cytoskeleton required for membrane ruffling.     

Guard cell cation channels are involved in Na+–induced stomatal closure in a halophyte

The halophyte Aster tripolium, unlike well-studied non-halophytic species, partially closes its stomata in response to high Na⁺ concentrations. Since A. tripolium possesses no specific morphological adaptation to salinity, this stomatal response, preventing excessive accumulation of Na⁺ within the shoot via control of the transpiration rate, is probably a principal feature of its salt tolerance within the shoot. The ionic basis of the stomatal response to Na⁺ was studied in guard cell protoplasts from A. tripolium and from a non-halophytic relative, Aster amellus, which exhibits classical stomatal opening on Na⁺. Patch-clamp studies revealed that plasma membrane K⁺ channels (inward and outward rectifiers) of the halophytic and the non-halophytic species are highly selective for K⁺ against Na⁺, and are very similar with respect to unitary conductance and direct sensitivity to Na⁺. On the other hand, both species possess a significant permeability to Na⁺ through non-rectifying cation channels activated by low (physiological) external Ca²⁺ concentrations. Finally, it appeared that the differential stomatal response between the two species is achieved, at least in part, by a Na⁺-sensing system in the halophyte which downregulates K⁺ uptake. Thus, increases in guard cell cytosolic Na⁺ concentration in A. tripolium but not in A. amellus, lead to a delayed (20–30 min) and dramatic deactivation of the K⁺ inward rectifier. This deactivation is probably mediated by an increase in cytosolic Ca²⁺ since buffering it abolishes the response. The possible role of K⁺ inward rectifiers in the response of A. tripolium’s stomata to Na⁺, suggested by patch-clamp studies, was confirmed by experiments demonstrating that specific blockade of inward rectifying channels mimics Na⁺ effects on stomatal aperture, and renders aperture refractory to Na⁺.     

CLE9 peptide‐induced stomatal closure is mediated by abscisic acid,hydrogen peroxide,and nitric oxide in Arabidopsis thaliana

CLE peptides have been implicated in various developmental processes of plants and mediate their responses to environmental stimuli. However, the biological relevance of most CLE genes remains to be functionally characterized. Here, we report that CLE9, which is expressed in stomata, acts as an essential regulator in the induction of stomatal closure. Exogenous application of CLE9 peptides or overexpression of CLE9 effectively led to stomatal closure and enhanced drought tolerance, whereas CLE9 loss‐of‐function mutants were sensitivity to drought stress. CLE9‐induced stomatal closure was impaired in abscisic acid (ABA)‐deficient mutants, indicating that ABA is required for CLE9‐medaited guard cell signalling. We further deciphered that two guard cell ABA‐signalling components, OST1 and SLAC1, were responsible for CLE9‐induced stomatal closure. MPK3 and MPK6 were activated by the CLE9 peptide, and CLE9 peptides failed to close stomata in mpk3 and mpk6 mutants. In addition, CLE9 peptides stimulated the induction of hydrogen peroxide (H₂O₂) and nitric oxide (NO) synthesis associated with stomatal closure, which was abolished in the NADPH oxidase‐deficient mutants or nitric reductase mutants, respectively. Collectively, our results reveal a novel ABA‐dependent function of CLE9 in the regulation of stomatal apertures, thereby suggesting a potential role of CLE9 in the stress acclimatization of plants.     

Phytochrome involvement in stomatal movement in Pisum sativum, Vicia faba and Pelargonium sp.

Red and blue light triggered the opening of isolated stomata of Pisum sativum L. cv. Peleg Alvador, Vicia faba L. (unknown cultivar) and Pelargonium sp. The stimulatory effect of short irradiation with red or blue light was reversed by a subsequent short irradiation with far-red light. In Pisum the stimulatory effect of a continuous irradiation with red or blue light was also abolished by a concomitant far-red light. In leaf pieces of P. sativum blue light was more effective than red, but not in isolated guard cells. In the presence of mesophyll, DCMU inhibited stomatal opening in red light more than in blue, and thus increased the relative response to blue light. This was less evident in isolated guard cells.     

The role of plasma membrane H+‐ATPase in jasmonate‐induced ion fluxes and stomatal closure in Arabidopsis thaliana

Methyl jasmonate (MeJA) elicits stomatal closure in many plant species. Stomatal closure is accompanied by large ion fluxes across the plasma membrane (PM). Here, we recorded the transmembrane ion fluxes of H⁺, Ca²⁺ and K⁺ in guard cells of wild‐type (Col‐0) Arabidopsis, the CORONATINE INSENSITIVE1 (COI1) mutant coi1‐1 and the PM H⁺‐ATPase mutants aha1‐6 and aha1‐7, using a non‐invasive micro‐test technique. We showed that MeJA induced transmembrane H⁺ efflux, Ca²⁺ influx and K⁺ efflux across the PM of Col‐0 guard cells. However, this ion transport was abolished in coi1‐1 guard cells, suggesting that MeJA‐induced transmembrane ion flux requires COI1. Furthermore, the H⁺ efflux and Ca²⁺ influx in Col‐0 guard cells was impaired by vanadate pre‐treatment or PM H⁺‐ATPase mutation, suggesting that the rapid H⁺ efflux mediated by PM H⁺‐ATPases could function upstream of the Ca²⁺ flux. After the rapid H⁺ efflux, the Col‐0 guard cells had a longer oscillation period than before MeJA treatment, indicating that the activity of the PM H⁺‐ATPase was reduced. Finally, the elevation of cytosolic Ca²⁺ concentration and the depolarized PM drive the efflux of K⁺ from the cell, resulting in loss of turgor and closure of the stomata.     

PLC-gamma1 signaling pathway and villin activation are involved in actin cytoskeleton reorganization induced by Na+/Pi cotransport up-regulation

BACKGROUND: The brief incubation of opossum kidney (OK) cells with low P(i) results in Na+/P(i) cotransport up-regulation and in substantial, but transient, cytoskeletal reorganization. In this study, we examined signaling events involved in the depolymerization of microfilaments. RESULTS: Confocal laser scanning microscopy, immunoblot and immunoprecipitation experiments revealed villin co-localization with mainly actin short filaments and monomers, indicating that under the conditions used, villin acted as an actin-severing protein. Further analysis revealed that low concentrations of extracellular phosphate resulted in phospholipase Cgammal (PLC-gammal) translocation to the actin cytoskeleton, without increases in its tyrosine phosphorylation. Additionally, tyrosine phosphorylation of a portion of insoluble villin was increased; whereas, only tyrosine phosphorylated villin associated with PLC-gammal. Although, tyrosine phosphorylation of PLC-gammal was not observed during Na+/P(i) cotransport up-regulation, genistein treatment abolished the enzyme's translocation to the actin cytoskeleton, as well as its association with villin. In addition, villin was found to associate with the 85-KDa subunit (p85) of phosphatidylinositol (PI)-3 kinase, concomitant with PLC-gammal, in the cytoskeletal fraction of Na+/P(i) cotransport up-regulated cells. CONCLUSIONS: Our observations suggest a signaling mechanism linking low ambient P(i) levels to the acute up-regulation of its cotransport with sodium and the depolymerization of the subcortical actin cytoskeleton.     

Ethylene mediates brassinosteroid‐induced stomatal closure via Gα protein‐activated hydrogen peroxide and nitric oxide production in Arabidopsis

Brassinosteroids (BRs) are essential for plant growth and development; however, whether and how they promote stomatal closure is not fully clear. In this study, we report that 24‐epibrassinolide (EBR), a bioactive BR, induces stomatal closure in Arabidopsis (Arabidopsis thaliana) by triggering a signal transduction pathway including ethylene synthesis, the activation of Gα protein, and hydrogen peroxide (H₂O₂) and nitric oxide (NO) production. EBR initiated a marked rise in ethylene, H₂O₂ and NO levels, necessary for stomatal closure in the wild type. These effects were abolished in mutant bri1‐301, and EBR failed to close the stomata of gpa1 mutants. Next, we found that both ethylene and Gα mediate the inductive effects of EBR on H₂O₂ and NO production. EBR‐triggered H₂O₂ and NO accumulation were canceled in the etr1 and gpa1 mutants, but were strengthened in the eto1‐1 mutant and the cGα line (constitutively overexpressing the G protein α‐subunit AtGPA1). Exogenously applied H₂O₂ or sodium nitroprusside (SNP) rescued the defects of etr1‐3 and gpa1 or etr1 and gpa1 mutants in EBR‐induced stomatal closure, whereas the stomata of eto1‐1/AtrbohF and cGα/AtrbohF or eto1‐1/nia1‐2 and cGα/nia1‐2 constructs had an analogous response to H₂O₂ or SNP as those of AtrbohF or Nia1‐2 mutants. Moreover, we provided evidence that Gα plays an important role in the responses of guard cells to ethylene. Gα activator CTX largely restored the lesion of the etr1‐3 mutant, but ethylene precursor ACC failed to rescue the defects of gpa1 mutants in EBR‐induced stomatal closure. Lastly, we demonstrated that Gα‐activated H₂O₂ production is required for NO synthesis. EBR failed to induce NO synthesis in mutant AtrbohF, but it led to H₂O₂ production in mutant Nia1‐2. Exogenously applied SNP rescued the defect of AtrbohF in EBR‐induced stomatal closure, but H₂O₂ did not reverse the lesion of EBR‐induced stomatal closure in Nia1‐2. Together, our results strongly suggest a signaling pathway in which EBR induces ethylene synthesis, thereby activating Gα, and then promotes AtrbohF‐dependent H₂O₂ production and subsequent Nia1‐catalyzed NO accumulation, and finally closes stomata.     

Copper amine oxidase and phospholipase D act independently in abscisic acid (ABA)-induced stomatal closure in Vicia faba and Arabidopsis

Recent evidence has demonstrated that both copper amine oxidase (CuAO; EC 1.4.3.6) and phospholipase D (PLD; EC 3.1.4.4) are involved in abscisic acid (ABA)-induced stomatal closure. In this study, we investigated the interaction between CuAO and PLD in the ABA response. Pretreatment with either CuAO or PLD inhibitors alone or that with both additively led to impairment of ABA-induced H₂O₂ production and stomatal closure in Vicia faba. ABA-stimulated PLD activation could not be inhibited by the CuAO inhibitor, and CuAO activity was not affected by the PLD inhibitor. These data suggest that CuAO and PLD act independently in the ABA response. To further examine PLD and CuAO activities in ABA responses, we used the Arabidopsis mutants cuaoζ and pldα1. Ablation of guard cell-expressed CuAOζ or PLDα1 gene retarded ABA-induced H₂O₂ generation and stomatal closure. As a product of PLD, phosphatidic acid (PA) substantially enhanced H₂O₂ production and stomatal closure in wide type, pldα1, and cuaoζ. Moreover, putrescine (Put), a substrate of CuAO as well as an activator of PLD, induced H₂O₂ production and stomatal closure in WT but not in both mutants. These results suggest that CuAO and PLD act independently in ABA-induced stomatal closure.     

Ethylene limits abscisic acid‐ or soil drying‐induced stomatal closure in aged wheat leaves

The mechanism of age‐induced decreased stomatal sensitivity to abscisic acid (ABA) and soil drying has been explored here. Older, fully expanded leaves partly lost their ability to close stomata in response to foliar ABA sprays, and soil drying which stimulated endogenous ABA production, while young fully expanded leaves closed their stomata more fully. However, ABA‐ or soil drying‐induced stomatal closure of older leaves was partly restored by pretreating plants with 1‐methylcyclopropene (1‐MCP), which can antagonize ethylene receptors, or by inoculating soil around the roots with the rhizobacterium Variovorax paradoxus 5C‐2, which contains 1‐aminocyclopropane‐1‐carboxylic acid (ACC)‐deaminase. ACC (the immediate biosynthetic precursor of ethylene) sprays revealed higher sensitivity of stomata to ethylene in older leaves than younger leaves, despite no differences in endogenous ACC concentrations or ethylene emission. Taken together, these results indicate that the relative insensitivity of stomatal closure to ABA and soil drying in older leaves is likely due to altered stomatal sensitivity to ethylene, rather than ethylene production. To our knowledge, this is the first study to mechanistically explain diminished stomatal responses to soil moisture deficit in older leaves, and the associated reduction in leaf water‐use efficiency.