Stomatal Closure In Vicia Faba Research Articles

The role of hydrogen sulfide and its relationship with hydrogen peroxide and nitric oxide in brassinosteroid-induced stomatal closure of Vicia faba L.

The role of hydrogen sulfide (H2S) and its relationship with hydrogen peroxide (H2O2) and nitric oxide (NO) in brassinosteroid-induced stomatal closure by using pharmacological, spectrophotographic and fluorescence microscope approaches in Vicia faba L. were investigated. In the present study, 2, 4-epibrassinolide (EBR, a bioactive BR) induced stomatal closure in a dose- and time-dependent manner, and the effects were inhibited by H2S modulators hypotaurine (HT), aminooxy acetic acid (AOA), hydroxylamine (NH2OH), potassium pyruvate (C3H3KO3) and ammonia (NH3), H2O2 modulators ascorbic acid (ASA), catalase (CAT), diphenylene iodonium(DPI) and salicylhydroxamic acid (SHAM), and NO modulators 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), NG-Nitro-L-arginine methyl ester, hydrochloride (L-NAME) and sodium tungstate (Na2WO4), respectively. Additionally, EBR caused increase of L-/D-cysteine desulfhydrase (L-/D-CDes) activity and H2S production in leaves, H2O2 and NO generation in guard cells, and the effects were significantly inhibited by H2S modulators, H2O2 modulators and NO modulators, respectively. Moreover, H2O2 modulators and NO modulators could significantly inhibit EBR-induced rise of L-/D-CDes activity and H2S production in the leaves of V. faba. However, H2S modulators failed to repress EBR-induced H2O2 and NO production in guard cells of V. faba. Together, our results suggested that H2S, H2O2 and NO probably participate in the signal transduction pathway of EBR-induced stomatal closure, and H2S may represent a novel component downstream of H2O2 and NO in EBR-induced stomatal closure of V. faba.

Hydrogen sulfide may function downstream of hydrogen peroxide in salt stress-induced stomatal closure in Vicia faba.

The roles of hydrogen sulfide (H2S) and hydrogen peroxide (H2O2) in signalling transduction of stomatal closure induced by salt stress were examined by using pharmacological, spectrophotographic and laser scanning confocal microscopic (LSCM) approaches in Vicia faba L. Salt stress resulted in stomatal closure, and this effect was blocked by H2S modulators hypotaurine (HT), aminooxy acetic acid (AOA), hydroxylamine (NH2OH), potassium pyruvate (C3H3KO3) and ammonia (NH3) and H2O2 modulators ascorbic acid (ASA), catalase (CAT), diphenylene iodonium (DPI). Additionally, salt stress induced H2S generation and increased L-/D-cysteine desulfhydrase (L-/D-CDes, pyridoxalphosphate-dependent enzyme) activity in leaves, and caused H2O2 production in guard cells, and these effects were significantly suppressed by H2S modulators and H2O2 modulators respectively. Moreover, H2O2 modulators suppressed salt stress-induced increase of H2S levels and L-/D-CDes activity in leaves as well as stomatal closure of V. faba. However, H2S modulators had no effects on salt stress-induced H2O2 production in guard cells. Altogether, our data suggested that H2S and H2O2 probably are involved in salt stress-induced stomatal closure, and H2S may function downstream of H2O2 in salt stress-induced stomatal movement in V. faba.

Hydrogen sulfide may function downstream of hydrogen peroxide in mediating darkness-induced stomatal closure in Vicia faba.

The relationship between hydrogen sulfide (H2S) and hydrogen peroxide (H2O2) during darkness-induced stomatal closure in Vicia faba L. was investigated by using pharmacological, spectrophotographic and lasers canning confocal microscopic approaches. Darkness-induced stomatal closure was inhibited by H2S scavenger hypotaurine (HT), H2S synthesis inhibitors aminooxy acetic acid (AOA) and hydroxylamine (NH2OH) and potassium pyruvate (N3H3KO3) and ammonia (NH3), which are the products of L-/D-cysteine desulfhydrase (L-/D-CDes). Moreover, darkness induced H2S generation and increased L-/D-CDes activity in leaves of V. faba. H2O2 scavenger and synthesis inhibitors suppressed darkness-induced increase of H2S levels and L-/D-CDes activity as well as stomatal closure in leaves of V. faba. However, H2S scavenger and synthesis inhibitors had no effect on darkness-induced H2O2 accumulation in guard cells of V. faba. From these data it can be deduced that H2S is involved in darkness-induced stomatal closure and acts downstream of H2O2 in V. faba.

Role of PI3P and Its Relationship with H2O2 or Nitric Oxide in Darkness-induced Stomatal Closure in Vicia faba

Role of PI3P and Its Relationship with H2O2 or Nitric Oxide in Darkness-induced Stomatal Closure in Vicia faba

Tyrosine phosphorylation mediates starch metabolism in guard cell of Vicia faba

AbstractProtein tyrosine phosphorylation plays a central role in many signaling pathways leading to cell growth and differentiation in animals. However, the cellular roles for tyrosine phosphorylation in plant remain unclear. Here we describe that tyrosine phosphorylation involved in stomata closing pathway. Genistein, an inhibitor of protein tyrosine kinases (PTKs), could prevent darkness and abscisic acid (ABA)-induced stomatal closure of Vicia faba L. Stomatal closure induced by high external Ca

Copper amine oxidase-catalysed hydrogen peroxide involves production of nitric oxide in darkness-induced stomatal closure in broad bean.

Hydrogen peroxide is an important intermediate in darkness-induced stomatal closure. In the present work, we provide evidence that copper amine oxidase (CuAO) was involved in H2O2 production in darkness-induced stomatal closure in Vicia faba L. Darkness activated CuAO in intercellular washing fluid from leaves. Aminoguanidine (AG) and 2-bromoethylamine (BEA), which were both irreversible inhibitors of CuAO, significantly suppressed darkness-induced stomatal closure and H2O2 generation. The effects of AG and BEA were reversed only by H2O2 but not by other products of CuAO. These results indicate that CuAO participates in darkness-induced stomatal closure through its reaction product, H2O2. Furthermore, darkness-induced nitric oxide (NO) production and cytosolic alkalinisation were obviously inhibited by AG and BEA, and only H2O2, among the products of CuAO, could reverse the effects, implying that the CuAO-catalysed product H2O2 is required for NO production and cytosolic alkalinisation to a large extent in darkness-induced stomatal closure. In addition, butyric acid blocked but methylamine enhanced the ability of H2O2 to reverse the effect of BEA on NO production, suggesting that cytosolic alkalinisation is involved in CuAO-mediated NO generation in darkness-induced stomatal closure.

Stomata prioritize their responses to multiple biotic and abiotic signal inputs.

Stomata are microscopic pores in leaf epidermis that regulate gas exchange between plants and the environment. Being natural openings on the leaf surface, stomata also serve as ports for the invasion of foliar pathogenic bacteria. Each stomatal pore is enclosed by a pair of guard cells that are able to sense a wide spectrum of biotic and abiotic stresses and respond by precisely adjusting the pore width. However, it is not clear whether stomatal responses to simultaneously imposed biotic and abiotic signals are mutually dependent on each other. Here we show that a genetically engineered Escherichia coli strain DH5α could trigger stomatal closure in Vicia faba, an innate immune response that might depend on NADPH oxidase-mediated ROS burst. DH5α-induced stomatal closure could be abolished or disguised under certain environmental conditions like low [CO2], darkness, and drought, etc. Foliar spraying of high concentrations of ABA could reduce stomatal aperture in high humidity-treated faba bean plants. Consistently, the aggressive multiplication of DH5α bacteria in Vicia faba leaves under high humidity could be alleviated by exogenous application of ABA. Our data suggest that a successful colonization of bacteria on the leaf surface is correlated with stomatal aperture regulation by a specific set of environmental factors.

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.

Involvement of copper amine oxidase (CuAO)-dependent hydrogen peroxide synthesis in ethylene-induced stomatal closure in Vicia faba

Ethylene promotes stomatal closure via inducing hydrogen peroxide (H2O2) generation. H2O2 can be catalytically synthesized by several enzymes in plants. Here, by means of stomatal bioassay, the analysis of enzyme activity and using laser-scanning confocal microscopy based on the H2O2-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA), the roles of copper amine oxidase (CuAO) in ethylene-induced H2O2 production in guard cells and stomatal closure in Vicia faba L. were investigated. 1-aminocyclopropane-1-carboxylic acid (ACC), an immediate precursor of ethylene synthesis, and ethylene gas significantly activated CuAO in intercellular washing fluid from leaves, the production of H2O2 in guard cells, and stomatal closure. These effects of ACC and ethylene gas were largely prevented by both aminoguanidine and 2-bromoethylamine, which are irreversible inhibitors of CuAO. Among major catalyzed and metabolized products of CuAO, only H2O2 could markedly promote stomatal closure and evidently reversed the effect of CuAO inhibitor on stomatal closure by ACC and ethylene gas. The data described above show that CuAO-mediated H2O2 production is involved in ethylene-induced stomatal closure.

Cytosolic alkalization-mediated H2O2 and NO production are involved in darkness-induced stomatal closure in Vicia faba

Ma, Y., She, X. and Yang, S. 2013. Cytosolic alkalization-mediated H2O2and NO production are involved in darkness-induced stomatal closure in Vicia faba. Can. J. Plant Sci. 93: 119-130. Darkness raised cytosolic pH, hydrogen peroxide (H2O2) and nitric oxide (NO) levels in guard cells while inducing Vicia faba stomatal closure. These darkness effects were prevented by weak acid butyric acid, H2O2 modulators ascorbic acid (ASA), catalase (CAT), diphenyleneiodonium (DPI) and NO modulators 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), NG-nitro-L-arg-methyl ester (L-NAME) respectively. The data suggest that cytosolic alkalization, H2O2 and NO all participate in darkness-induced stomatal closure. During darkness treatment, pH rise became noticeable at 10 min and peaked at 25 min, while H2O2 and NO production increased significantly at 20 min and reached their maximums at 40 min. The H2O2 and NO levels were increased by methylamine in light and decreased by butyric acid in darkness....

Cytosolic alkalization-mediated H2O2 and NO production are involved in darkness-induced stomatal closure in Vicia faba

Ma, Y., She, X. and Yang, S. 2013. Cytosolic alkalization-mediated H 2 O 2 and NO production are involved in darkness-induced stomatal closure in Vicia faba. Can. J. Plant Sci. 93: 119–130. Darkness raised cytosolic pH, hydrogen peroxide (H2O2) and nitric oxide (NO) levels in guard cells while inducing Vicia faba stomatal closure. These darkness effects were prevented by weak acid butyric acid, H2O2 modulators ascorbic acid (ASA), catalase (CAT), diphenyleneiodonium (DPI) and NO modulators 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), NG-nitro-L-arg-methyl ester (L-NAME) respectively. The data suggest that cytosolic alkalization, H2O2 and NO all participate in darkness-induced stomatal closure. During darkness treatment, pH rise became noticeable at 10 min and peaked at 25 min, while H2O2 and NO production increased significantly at 20 min and reached their maximums at 40 min. The H2O2 and NO levels were increased by methylamine in light and decreased by butyric acid in darkness. The results show that cytosolic alkalization induces H2O2 and NO production. ASA, CAT and DPI suppressed NO production by methylamine, c-PTIO and L-NAME prevented H2O2 generation by methylamine. Calcium chelator 1,2-bis (2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) restricted darkness-induced alkalization, H2O2 and NO production and stomatal closure. We suggest that cytosolic alkalization is necessary for H2O2 and NO production during darkness-induced stomatal closure. H2O2 mediates NO synthesis by alkalization, and vice versa. Calcium may act upstream of cytosolic alkalization, H2O2 and NO production, besides its known action downstream of H2O2 and NO.

Hydrogen Sulfide May Function Downstream of Nitric Oxide in Ethylene-Induced Stomatal Closure in Vicia faba L.

Pharmacological, laser scanning confocal microscopic (LSCM), and spectrophotographic approaches were used to study the roles of hydrogen sulfide (H2S) and nitric oxide (NO) in signaling transduction of stomatal movement in response to ethylene in Vicia faba L. Ethylene treatment resulted in the dose-dependent stomatal closure under light, and this effect was blocked by the inhibitors of H2S biosynthesis in V. faba L. Additionally, ethylene induces H2S generation and increases L-/D-cysteine desulfhydrase (pyridoxalphosphate-dependent enzyme) activity in leaves of V. faba L. Inhibitors of H2S biosynthesis have no effect on the ethylene-induced stomatal closure, NO accumulation, and nitrate reductase (NR) activity in guard cells or leaves of V. faba L. Moreover, the ethylene-induced increase of H2S levels and L-/D-cysteine desulfhydrase activity declined when NO generation was inhibited. Therefore, we conclude that H2S and NO probably are involved in the signal transduction pathway of ethylene-induced stomatal closure. H2S may represent a novel component downstream of NO in the ethylene-induced stomatal movement in V. faba L.

Ethylene inhibits abscisic acid-induced stomatal closure in Vicia faba via reducing nitric oxide levels in guard cells

The relationship between nitric oxide (NO) reduction and the inhibition of abscisic acid (ABA)-induced stomatal closure by ethylene was analysed. ABA treatment induced NO production and stomatal closure, but ethylene inhibited the effects of ABA on these two parameters. Like 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a specific scavenger of NO, haemoglobin (Hb), the potent scavenger of NO/carbon monoxide (CO) and N G -nitro-L-Arg-methyl ester (L-NAME), an inhibitor of animal nitric oxide synthases (NOS; EC 1.14.13.39), both the ethylene-releasing compound 2-chloroethylene phosphonic acid (ethephon, ETH) and 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, were found to inhibit stomatal closure by ABA and to reduce NO levels by ABA in guard cells, indicating that the ethylene-caused inhibition of ABA-induced stomatal closure involves a decrease in NO levels in guard cells. In addition, and similar to cPTIO and Hb, ACC/ETH suppressed sodium nitroprusside (SNP)-induced stomatal closure and NO levels in guard cells treated with SNP in the light. ACC/ETH also reopened stomata that had been closed by ABA and reduced NO levels that had been generated by ABA, showing that ethylene causes NO removal in guard cells. However, the above-mentioned effect of ACC/ETH was dissimilar to that of L-NAME, which was incapable of reducing NO levels by SNP or abolishing NO that had been generated by ABA. We suggest that ethylene probably induces NO removal, thereby reducing NO levels in Vicia faba guard cells, and finally inhibits stomatal closure induced by ABA. Furthermore, the pattern of NO reduction caused by ethylene is also discussed.

Excretion and folding of plasmalemma function to accommodate alterations in guard cell volume during stomatal closure in Vicia faba L.

Stomatal movement results in large and repetitive changes in cell volume and consequently surface area. While endocytosis has been extensively studied and is thought to be a major mechanism for accommodating the volume changes as evidenced mainly by fluorescent labelling and confocal imaging, studies at the ultrastructural level in intact guard cells of stomata regulated by natural factors have never been reported. Here, it is reported that excretion and folding of the plasmalemma are critical for accommodating the volume alterations in intact guard cells in Vicia faba L. Using transmission electron microscopy in combination with laser confocal microscopy, it was observed that in fully opened stomata the plasmalemma was smooth and tightly adhered to the cell walls while a whole large vacuole appeared in the cell. In the closed stomata, besides vacuole fragmentation, endocytosis of the tonoplast rather than the plasmalemma commonly occurred. Importantly, in stomata where pore closure was induced by circadian rhythm or CO2, numerous tiny vesicles were found outside the plasmalemma and, moreover, extensive folding of the plasmalemma could also be found in some regions of the cells. Additionally, an unknown structure was found at the interface between the plasmalemma and cell walls, especially in those areas of the cell where extensive folding occurred, suggesting that plasmalemma turnover is possibly associated with an interaction between the plasmalemma and cell walls. Collectively, the results strongly indicate that excretion and folding of the plasmalemma are critical for the accommodation of the cell volume alterations during stomatal movement.

Phosphatidic Acid Inhibits Blue Light-Induced Stomatal Opening via Inhibition of Protein Phosphatase 1

Stomata open in response to blue light under a background of red light. The plant hormone abscisic acid (ABA) inhibits blue light-dependent stomatal opening, an effect essential for promoting stomatal closure in the daytime to prevent water loss. However, the mechanisms and molecular targets of this inhibition in the blue light signaling pathway remain unknown. Here, we report that phosphatidic acid (PA), a phospholipid second messenger produced by ABA in guard cells, inhibits protein phosphatase 1 (PP1), a positive regulator of blue light signaling, and PA plays a role in stimulating stomatal closure in Vicia faba. Biochemical analysis revealed that PA directly inhibited the phosphatase activity of the catalytic subunit of V. faba PP1 (PP1c) in vitro. PA inhibited blue light-dependent stomatal opening but did not affect red light- or fusicoccin-induced stomatal opening. PA also inhibited blue light-dependent H(+) pumping and phosphorylation of the plasma membrane H(+)-ATPase. However, PA did not inhibit the autophosphorylation of phototropins, blue light receptors for stomatal opening. Furthermore, 1-butanol, a selective inhibitor of phospholipase D, which produces PA via hydrolysis of phospholipids, diminished the ABA-induced inhibition of blue light-dependent stomatal opening and H(+) pumping. We also show that hydrogen peroxide and nitric oxide, which are intermediates in ABA signaling, inhibited the blue light responses of stomata and that 1-butanol diminished these inhibitions. From these results, we conclude that PA inhibits blue light signaling in guard cells by PP1c inhibition, accelerating stomatal closure, and that PP1 is a cross talk point between blue light and ABA signaling pathways in guard cells.

MEK1/2 and p38-like MAP kinase successively mediate H2O2 signaling in Vicia guard cell

As a second messenger, H2O2 generation and signal transduction is subtly controlled and involves various signal elements, among which the members of MAP kinase family. The increasing evdences indicated that both MEK1/2 and p38-like MAP protein kinase have been shown to mediate ABA-induced H2O2 signaling in plant cells. Here we analyze the mechanisms of similarity and difference between MEK1/2 and p38-like MAP protein kinase in mediating ABA-induced H2O2 generation, inhibition of inward K+ currents, and stomatal closure, suggesting that activation of MEK1/2 is prior to p38-like protein kinase in Vicia guard cells.Addendum to: Jing J, Peng-Tao W, Guo-Yong A, Peng-Cheng W, Chun-Peng S. The involvement of a P38-like MAP kinase in ABA-induced and H2O2-mediated stomatal closure in Vicia faba L. Plant Cell Rep 2008; 27:377-85, DOI: 10.1007/s00299-007-0449-x.

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

H(2)O(2) is an essential signal in absicic acid (ABA)-induced stomatal closure. It can be synthesized by several enzymes in plants. In this study, the roles of copper amine oxidase (CuAO) in H(2)O(2) production and stomatal closure were investigated. Exogenous ABA stimulated apoplast CuAO activity, increased H(2)O(2) production and [Ca(2+)](cyt) levels in Vicia faba guard cells, and induced stomatal closure. These processes were impaired by CuAO inhibitor(s). In the metabolized products of CuAO, only H(2)O(2) could induce stomatal closure. By the analysis of enzyme kinetics and polyamine contents in leaves, putrescine was regarded as a substrate of CuAO. Putrescine showed similar effects with ABA on the regulation of H(2)O(2) production, [Ca(2+)](cyt) levels, as well as stomatal closure. The results suggest that CuAO in V. faba guard cells is an essential enzymatic source for H(2)O(2) production in ABA-induced stomatal closure via the degradation of putrescine. Calcium messenger is an important intermediate in this process.

Transient expression of AtNCED3 and AAO3 genes in guard cells causes stomatal closure in Vicia faba

Abscisic acid (ABA) regulates stomatal closure in response to water loss. Here, we examined the competence of guard cells to synthesize ABA, using two Arabidopsis ABA biosynthetic enzymes. 35S pro::AtNCED3-GFP and AAO3-GFP were introduced into guard cells of broad bean leaves. AtNCED3-GFP expression was detected at the chloroplasts, whereas green fluorescent protein (GFP) and AAO3-GFP were in the cytosol. The stomatal aperture was decreased in AtNCED3-GFP- and AAO3-GFP-transformed guard cells. This indicated that ABA biosynthesis is stimulated by heterologous expression of AtNCED3 and Arabidopsis aldehyde oxidase 3 (AAO3) proteins, which both seem to be regulatory enzymes for ABA biosynthesis in these cells. Furthermore, stomatal closure by the expression of AtNCED3 and AAO3 suggested that the substrates of the enzymes are present and native ABA-biosynthesis enzymes are active in guard cells.

The involvement of a P38-like MAP kinase in ABA-induced and H2O2-mediated stomatal closure in Vicia faba L.

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.

Involvement of carbon monoxide produced by heme oxygenase in ABA-induced stomatal closure in Vicia faba and its proposed signal transduction pathway

Carbon monoxide (CO) has recently proven to be an important bioactive or signaling molecule in mammalian cells. Its effects are mainly mediated by nitric oxide (NO) and cyclic GMP (cGMP). In Vicia faba leaves, CO production and heme oxygenase (HO) activity, an important CO synthetic enzyme, are first reported to increase in response to ABA treatment, which could result in stomatal closure. Interestingly, ABA-induced stomatal closure in V. faba guard cells is partially blocked when the synthetic CO inhibitor ZnPP, or the CO/NO scavenger Hb is added. Furthermore, we show that, exogenously applied CO donor, hematin, and CO aqueous solution not only result in the enhancement of CO release, but also time-dependently induce stomatal closure, and the latter is mimicked by the application of an NO donor SNP. The above-mentioned stomatal closure effects are differentially reversed by the addition of tungstate, a potent inhibitor of NO synthetic enzyme nitrate reductase (NR), the specific NO scavenger cPTIO, ZnPP, or Hb. During treatment for 4 h, SNP, 0.01% CO aqueous solution or hematin significantly triggers NO synthesis, whereas cPTIO, or tungstate approximately fully inhibits NO fluorescence. Additionally, application of the GC inhibitor ODQ blocks CO-induced stomatal closure. This inhibition could be reversed when 8-Br-cGMP is added. Thus, the above results suggest that CO produced by HO is involved in ABA-induced stomatal closure, and NO and cGMP may function as downstream intermediates in the CO signaling responsible for stomatal closure.