ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) are key signalling molecules produced in response to various stimuli and involved in a diverse range of plant signal transduction processes. Nitric oxide and H(2)O(2) have been identified as essential components of the complex signalling network inducing stomatal closure in response to the phytohormone abscisic acid (ABA). A close inter-relationship exists between ABA and the spatial and temporal production and action of both NO and H(2)O(2) in guard cells. This study shows that, in Arabidopsis thaliana guard cells, ABA-mediated NO generation is in fact dependent on ABA-induced H(2)O(2) production. Stomatal closure induced by H(2)O(2) is inhibited by the removal of NO with NO scavenger, and both ABA and H(2)O(2) stimulate guard cell NO synthesis. Conversely, NO-induced stomatal closure does not require H(2)O(2) synthesis nor does NO treatment induce H(2)O(2) production in guard cells. Tungstate inhibition of the NO-generating enzyme nitrate reductase (NR) attenuates NO production in response to nitrite in vitro and in response to H(2)O(2) and ABA in vivo. Genetic data demonstrate that NR is the major source of NO in guard cells in response to ABA-mediated H(2)O(2) synthesis. In the NR double mutant nia1, nia2 both ABA and H(2)O(2) fail to induce NO production or stomatal closure, but in the nitric oxide synthase deficient Atnos1 mutant, responses to H(2)O(2) are not impaired. Importantly, we show that in the NADPH oxidase deficient double mutant atrbohD/F, NO synthesis and stomatal closure to ABA are severely reduced, indicating that endogenous H(2)O(2) production induced by ABA is required for NO synthesis. In summary, our physiological and genetic data demonstrate a strong inter-relationship between ABA, endogenous H(2)O(2) and NO-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     

Guard cell-specific inhibition of Arabidopsis MPK3 expression causes abnormal stomatal responses to abscisic acid and hydrogen peroxide

MAP kinases have been linked to guard cell signalling. Arabidopsis thaliana MAP Kinase 3 (MPK3) is known to be activated by abscisic acid (ABA) and hydrogen peroxide (H(2)O(2)), which also control stomatal movements. We therefore studied the possible role of MPK3 in guard cell signalling through guard cell-specific antisense inhibition of MPK3 expression. Such transgenic plants contained reduced levels of MPK3 mRNA in the guard cells and displayed partial insensitivity to ABA in inhibition of stomatal opening, but responded normally to this hormone in stomatal closure. However, ABA-induced stomatal closure was reduced compared with controls when cytoplasmic alkalinization was prevented with sodium butyrate. MPK3 antisense plants were less sensitive to exogenous H(2)O(2), both in inhibition of stomatal opening and in promotion of stomatal closure, thus MPK3 is required for the signalling of this compound. ABA-induced H(2)O(2) synthesis was normal in these plants, indicating that MPK3 probably acts in signalling downstream of H(2)O(2). These results provide clear evidence for the important role of MPK3 in the perception of ABA and H(2)O(2) in guard cells.     

NO可能作为H2O2的下游信号介导ABA诱导的蚕豆气孔关闭

ABA、H2O2和硝普钠(SNP)均能诱导蚕豆气孔关闭.NO的清除剂c-PTIO可以减轻由ABA或H2O2所诱导的蚕豆气孔关闭的程度,而过氧化氢酶(CAT)则不能减轻NO诱导的气孔关闭程度.激光共聚焦显微检测结果显示,10μmo1/L的ABA处理后,胞内H2O2的产生速率明显高于NO的产生速率;CAT几乎可完全抑制ABA所诱导的DAF的荧光增加;外源H2O2能显著诱导胞内DAF的荧光增加;c-PTIO对ABA诱导的DCF荧光略有促进作用,但外源SNP不能诱导胞内DCF荧光增加.这些结果表明,在ABA诱导气孔关闭过程中,H2O2可能在NO的上游起作用并受NO的负反馈调节.     

ABA, hydrogen peroxide and nitric oxide signalling in stomatal guard cells

Increased synthesis and redistribution of the phytohormone abscisic acid (ABA) in response to water deficit stress initiates an intricate network of signalling pathways in guard cells leading to stomatal closure. Despite the large number of ABA signalling intermediates that are known in guard cells, new discoveries are still being made. Recently, the reactive oxygen species hydrogen peroxide (H2O2) and the reactive nitrogen species nitric oxide (NO) have been identified as key molecules regulating ABA-induced stomatal closure in various species. As with many other physiological responses in which H2O2 and NO are involved, stomatal closure in response to ABA also appears to require the tandem synthesis and action of both these signalling molecules. Recent pharmacological and genetic data have identified NADPH oxidase as a source of H2O2, whilst nitrate reductase has been identified as a source of NO in Arabidopsis guard cells. Some signalling components positioned downstream of H2O2 and NO are calcium, protein kinases and cyclic GMP. However, the exact interaction between the various signalling components in response to H2O2 and NO in guard cells remains to be established.     

NO对He-Ne激光和增强UV-B辐射小麦幼苗气孔运动的作用机理研究

为探讨NO对He-Ne激光和增强UV-B辐射小麦（Triticum aestivuml）气孔运动的作用机理,采用低剂量（5 mW.mm-2）He-Ne激光和增强（10.08 kJ.m-2.d-1）UV-B辐射并结合药理学实验和激光共聚焦显微技术,对ML7113小麦的叶片及表皮条进行不同的处理,结果显示：（1）UV-B辐射既可诱导小麦叶片气孔关闭,又能够明显增加气孔保卫细胞和叶片的NO水平,且NO清除剂明显抑制了UV-B辐射诱导的小麦叶片气孔关闭,同时气孔保卫细胞和叶片内的NO含量明显减少。（2）一氧化氮合酶（NOS）抑制剂L-NAME对经UV-B辐射诱导的小麦幼苗气孔开度及保卫细胞和叶片内NO含量的抑制程度明显大于硝酸还原酶（NR）抑制剂NaN3对其的抑制程度,说明一氧化氮合酶（NOS）合成途径是小麦叶片经UV-B辐射后NO的主要产生途径。（3）就气孔开度而言,L〉CK〉BL〉B。就小麦叶片及保卫细胞内NO含量而言,B〉BL〉CK〉L。就硝酸还原酶（NR）和一氧化氮合酶（NOS）的活性而言,B组NR活性最低,NOS活性最高,L组NR活性最高,NOS活性最低。表明经He-Ne激光和增强UV-B辐射诱导的小麦气孔开度的变化确实与保卫细胞及叶片中NO含量的多少有关,气孔开度的减小及增大对应于NO含量的增多或减少,同时进一步证实了小麦叶片经He-Ne激光单独辐照后,NO的主要合成途径也来源于NOS途径。     

Cytosolic acidification precedes nitric oxide removal during inhibition of ABA-induced stomatal closure by fusicoccin

The role of nitric oxide (NO) and the relationship between NO and cytosolic pH during inhibition of ABA effect by fusicoccin (FC) in guard cells of Vicia faba were analyzed. ABA induced NO generation and stomatal closure, but FC inhibited the effects of ABA. Treatment with 2-(4-carboxyphenyl)-4,4,5,5-tetra-methylimidazoline-1-oxyl-3-oxide (cPTIO) and N^G-nitro-L-Arg-methyl ester (L-NAME) mimicked the effects of FC. These data suggest that inhibition of ABA effect by FC is possibly related to the decreasing in the NO level. Furthermore, like cPTIO, FC not only suppressed stomatal closure and NO level in guard cells treated with NO donor sodium nitroprusside (SNP), but also reopened stomata, which had been closed by ABA, and reduced the level of NO in guard cells that had been produced by ABA, indicating that FC caused NO removal. Butyric acid simulated the effects of FC on the stomatal aperture and increased NO levels in guard cells treated with SNP and had been closed by ABA, and both FC and butyric acid surely reduced cytosolic pH, which demonstrates that cytosolic acidification mediates FC-induced NO removal. Taken together, our results show that FC induces NO removal and reduces NO level via cytosolic acidification in guard cells, thus inhibiting ABA effect.     

Cyclic adenosine 5′‐diphosphoribose (cADPR) cyclic guanosine 3′,5′‐monophosphate positively function in Ca2+ elevation in methyl jasmonate‐induced stomatal closure,cADPR is required for methyl jasmonate‐induced ROS accumulation NO production in guard cells

Methyl jasmonate (MeJA) signalling shares several signal components with abscisic acid (ABA) signalling in guard cells. Cyclic adenosine 5′‐diphosphoribose (cADPR) and cyclic guanosine 3′,5′‐monophosphate (cGMP) are second messengers in ABA‐induced stomatal closure. In order to clarify involvement of cADPR and cGMP in MeJA‐induced stomatal closure in Arabidopsis thaliana (Col‐0), we investigated effects of an inhibitor of cADPR synthesis, nicotinamide (NA), and an inhibitor of cGMP synthesis, LY83583 (LY, 6‐anilino‐5,8‐quinolinedione), on MeJA‐induced stomatal closure. Treatment with NA and LY inhibited MeJA‐induced stomatal closure. NA inhibited MeJA‐induced reactive oxygen species (ROS) accumulation and nitric oxide (NO) production in guard cells. NA and LY suppressed transient elevations elicited by MeJA in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) in guard cells. These results suggest that cADPR and cGMP positively function in [Ca²⁺]_cyt elevation in MeJA‐induced stomatal closure, are signalling components shared with ABA‐induced stomatal closure in Arabidopsis, and that cADPR is required for MeJA‐induced ROS accumulation and NO production in Arabidopsis guard cells.     

Nitric oxide is a signaling intermediate during bicarbonate-induced stomatal closure in Pisum sativum

The role of nitric oxide (NO) during bicarbonate-induced stomatal closure was studied in the abaxial epidermis of Pisum sativum . A few experiments were done with 10 μ M ABA, for comparison. The presence of 2 m M sodium bicarbonate or 10 μ M ABA induced an increase of NO in guard cells. Elevation of NO by sodium nitroprusside induced stomatal closure and enhanced further the closure by bicarbonate. The bicarbonate induced increase in NO of guard cells, or stomatal closure was prevented partially by 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide, an NO scavenger and N -nitro- l -Arg-methyl ester, an inhibitor of NO synthase (NOS). These results suggested that guard cells generated NO on exposure to bicarbonate and that NOS was involved at least partially in such NO production. Time course experiments revealed that on exposure to bicarbonate or ABA, the rise in guard cell NO production peaked within 10 min. Experiments using pharmacological compounds like wortmannin/LY294002 (phosphatidylinositol 3 kinase inhibitors), 1 H -(1,2,4)-oxadiazole-[4,3 a ]quinoxalin-1-one (guanylyl cyclase inhibitor), nicotinamide (cyclic adenosine diphosphate ribose antagonist), guanosine 5'-O-(2-thiodiphosphate) (G-protein antagonist) suggested a role of phosphatidylinositol 3-phosphate or G-proteins during bicarbonate-induced stomatal closure.     

FIA functions as an early signal component of abscisic acid signal cascade in Vicia faba guard cells

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.     

Nitric oxide production occurs after cytosolic alkalinization during stomatal closure induced by abscisic acid

Abscisic acid (ABA) raised the cytosolic pH and nitric oxide (NO) levels in guard cells while inducing stomatal closure in epidermis of Pisum sativum. Butyrate (a weak acid) reduced the cytosolic pH/NO production and prevented stomatal closure by ABA. Methylamine (a weak base) enhanced the cytosolic alkalinization and aggravated stomatal closure by ABA. The rise in guard cell pH because of ABA became noticeable after 6 min and peaked at 12 min, while NO production started at 9 min and peaked at 18 min. These results suggested that NO production was downstream of the rise in cytosolic pH. The ABA-induced increase in NO of guard cells and stomatal closure was prevented by 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide (cPTIO, a NO scavenger) and partially by N-nitro-L-Arg-methyl ester (L-NAME, an inhibitor of NO synthase). In contrast, cPTIO or L-NAME had only a marginal effect on the pH rise induced by ABA. Ethylene glycol tetraacetic acid (EGTA, a calcium chelator) prevented ABA-induced stomatal closure while restricting cytosolic pH rise and NO production. We suggest that during ABA-induced stomatal closure, a rise in cytosolic pH is necessary for NO production. Calcium may act upstream of cytosolic alkalinization and NO production, besides its known function as a downstream component.     

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.     

Reactive oxygen species and nitric oxide are involved in ABA inhibition of stomatal opening

Although nitric oxide (NO) and reactive oxygen species (ROS) are essential signalling molecules required for mediation of abscisic acid (ABA)-induced stomatal closure, it is not known whether these molecules also mediate the ABA inhibition of stomatal opening. In this study, we investigated the role of NO and ROS in the ABA inhibition of stomatal opening in Vicia faba. ABA induced both NO and ROS synthesis, and the NO scavenger reduced the ABA inhibition of stomatal opening. Exogenous NO and hydrogen peroxide (H2O2) also inhibited stomatal opening, indicating that NO and ROS are involved in the inhibition signalling process. An inhibitor of nitric oxide synthase (NOS) reversed the ABA inhibition of stomatal opening. Either the NO scavenger or the NOS inhibitor also reversed the process in the H2O2 inhibition of stomatal opening. We found that in the ABA inhibition of stomatal opening, NO is downstream of ROS in the signalling process, and NO is synthesized by a NOS-like enzyme.     

cGMP-dependent ABA-induced stomatal closure in the ABA-insensitive Arabidopsis mutant abi1-1

? The drought hormone abscisic acid (ABA) is widely known to produce reductions in stomatal aperture in guard cells. The second messenger cyclic guanosine 3', 5'-monophosphate (cGMP) is thought to form part of the signalling pathway by which ABA induces stomatal closure. ? We have examined the signalling events during cGMP-dependent ABA-induced stomatal closure in wild-type Arabidopsis plants and plants of the ABA-insensitive Arabidopsis mutant abi1-1. ? We show that cGMP acts downstream of hydrogen peroxide (H(2) O(2) ) and nitric oxide (NO) in the signalling pathway by which ABA induces stomatal closure. H(2) O(2) - and NO-induced increases in the cytosolic free calcium concentration ([Ca(2+) ](cyt) ) were cGMP-dependent, positioning cGMP upstream of [Ca(2+) ](cyt) , and involved the action of the type 2C protein phosphatase ABI1. Increases in cGMP were mediated through the stimulation of guanylyl cyclase by H(2) O(2) and NO. We identify nucleoside diphosphate kinase as a new cGMP target protein in Arabidopsis. ? This study positions cGMP downstream of ABA-induced changes in H(2) O(2) and NO, and upstream of increases in [Ca(2+) ](cyt) in the signalling pathway leading to stomatal closure.     

Pyrabactin, an ABA agonist, induced stomatal closure and changes in signalling components of guard cells in abaxial epidermis of Pisum sativum

Pyrabactin, a synthetic agonist of abscisic acid (ABA), inhibits seed germination and hypocotyl growth and stimulates gene expression in a very similar way to ABA, implying the possible modulation of stomatal function by pyrabactin as well. The effect of pyrabactin on stomatal closure and secondary messengers was therefore studied in guard cells of Pisum sativum abaxial epidermis. Pyrabactin caused marked stomatal closure in a pattern similar to ABA. In addition, pyrabactin elevated the levels of reactive oxygen species (ROS), nitric oxide (NO), and cytoplasmic pH levels in guard cells, as indicated by the respective fluorophores. However, apyrabactin, an inactive analogue of ABA, did not affect either stomatal closure or the signalling components of guard cells. The effects of pyrabactin-induced changes were reversed by pharmalogical compounds that modulate ROS, NO or cytoplasmic pH levels, quite similar to ABA effects. Fusicoccin, a fungal toxin, could reverse the stomatal closure caused by pyrabactin, as well as that caused by ABA. Experiments on stomatal closure by varying concentrations of ABA, in the presence of fixed concentration of pyrabactin, and vice versa, revealed that the actions of ABA and pyrabactin were additive. Further kinetic analysis of data revealed that the apparent K(D) of ABA was increased almost 4-fold in the presence of ABA, suggesting that pyrabactin and ABA were competing with each other either at the same site or close to the active site. It is proposed that pyrabactin could be used to examine the ABA-related signal-transduction components in stomatal guard cells as well as in other plant tissues. It is also suggested that pyrabactin can be used as an antitranspirant or as a priming agent for improving the drought tolerance of crop plants.     

Phospholipase Dδ is involved in nitric oxide-induced stomatal closure

Nitric oxide (NO) has recently emerged as a second messenger involved in the complex network of signaling events that regulate stomatal closure. Little is known about the signaling events occurring downstream of NO. Previously, we demonstrated the involvement of phospholipase D (PLD) in NO signaling during stomatal closure. PLDδ, one of the 12 Arabidopsis PLDs, is involved in dehydration stress responses. To investigate the role of PLDδ in NO signaling in guard cells, we analyzed guard cells responses using Arabidopsis wild type and two independent pldδ single mutants. In this work, we show that pldδ mutants failed to close the stomata in response to NO. Treatments with phosphatidic acid, the product of PLD activity, induced stomatal closure in pldδ mutants. Abscisic acid (ABA) signaling in guard cells involved H₂O₂ and NO production, both required for ABA-induced stomatal closure. pldδ guard cells produced similar NO and H₂O₂ levels as the wild type in response to ABA. However, ABA- or H₂O₂-induced stomatal closure was impaired in pldδ plants. These data indicate that PLDδ is downstream of NO and H₂O₂ in ABA-induced stomatal closure.     

Movement of Abscisic Acid into the Apoplast in Response to Water Stress in Xanthium strumarium L

The effect of water stress on the redistribution of abcisic acid (ABA) in mature leaves of Xanthium strumarium L. was investigated using a pressure dehydration technique. In both turgid and stressed leaves, the ABA in the xylem exudate, the `apoplastic' ABA, increased before `bulk leaf' stress-induced ABA accumulation began. In the initially turgid leaves, the ABA level remained constant in both the apoplast and the leaf as a whole until wilting symptoms appeared. Following turgor loss, sufficient quantities of ABA moved into the apoplast to stimulate stomatal closure. Thus, the initial increase of apoplastic ABA may be relevant to the rapid stomatal closure seen in stressed leaves before their bulk leaf ABA levels rise.

Following recovery from water stress, elevated levels of ABA remained in the apoplast after the bulk leaf contents had returned to their prestress values. This apoplastic ABA may retard stomatal reopening during the initial recovery period.

     

Calcium ions as second messengers in guard cell signal transduction

Ca²⁺ is a ubiquitous second messenger in plant cell signalling. In this review we consider the role of Ca²⁺-based signal transduction in stomatal guard cells focusing on three important areas: (1) the regulation of guard cell turgor relations and the control of gene expression in guard cells, (2) the control of specificity in Ca²⁺ signalling, (3) emerging technologies and new approaches for studying intracellular signalling. Stomatal apertures alter in response to a wide array of environmental stimuli as a result of changes in guard cell turgor. For example, the plant hormone abscisic acid (ABA) stimulates a reduction in stomatal aperture through a decrease in guard cell turgor. Furthermore, guard cells have been shown to be competent to relay an ABA signal from its site of perception to the nucleus. An increase in the concentration of cytosolic free Ca²⁺ ([Ca²⁺]₁) is central to the mechanisms underlying ABA-induced changes in guard cell turgor. We describe a possible model of Ca²⁺-based ABA signal transduction during stomatal closure and discuss recent evidence which suggests that Ca²⁺ is also involved in ABA nuclear signal transduction. Many other environmental stimuli which affect stomatal apertures, in addition to ABA, induce an increase in guard cell [Ca²⁺]₁) This raises questions regarding how increases in [Ca²⁺]₁) can be a common component in the signal transduction pathways by which stimuli cause both stomatal opening and closure. We discuss several mechanisms of increasing the amount of information contained within the Ca²⁺ signal, including encoding information in a stimulus-specific Ca²⁺ signal or Ca²⁺ signature', the concept of the ‘physiological address’ of the cell, and the use of other second messengers. We conclude by addressing the emerging technologies and new approaches which can be used in conjunction with guard cells to dissect further the molecular mechanisms of Ca²⁺-mediated signalling in plants.     

Abscisic acid (ABA) inhibits light-induced stomatal opening through calcium- and nitric oxide-mediated signaling pathways

Nitric oxide (NO) is an important signaling component of ABA-induced stomatal closure. However, only fragmentary data are available about NO effect on the inhibition of stomatal opening. Here, we present results supporting that, in Vicia faba guard cells, there is a critical Ca2+-dependent NO increase required for the ABA-mediated inhibition of stomatal opening. Light-induced stomatal opening was inhibited by exogenous NO in V. faba epidermal strips. Furthermore, ABA-mediated inhibition of stomatal opening was blocked by the specific NO scavenger cPTIO, supporting the involvement of endogenous NO in this process. Since the raise in Ca2+ concentration is a pre-requisite in ABA-mediated inhibition of stomatal opening, it was interesting to establish how does Ca2+, NO and ABA interact in the inhibition of light-induced stomatal opening. The permeable Ca2+ specific buffer BAPTA-AM blocked both ABA- and Ca2+- but not NO-mediated inhibition of stomatal opening. The NO synthase (NOS) specific inhibitor L-NAME prevented Ca2+-mediated inhibition of stomatal opening, indicating that a NOS-like activity was required for Ca2+ signaling. Furthermore, experiments using the NO specific fluorescent probe DAF-2DA indicated that Ca2+ induces an increase of endogenous NO. These results indicate that, in addition to the roles in ABA-triggered stomatal closure, both NO and Ca2+ are active components of signaling events acting in ABA inhibition of light-induced stomatal opening. Results also support that Ca2+ induces the NO production through the activation of a NOS-like activity.