Interaction of intracellular hydrogen peroxide accumulation with nitric oxide production in abscisic acid signaling in guard cells.

Reactive oxygen species and nitric oxide (NO•) concomitantly play essential roles in guard cell signaling. Studies using catalase mutants have revealed that the inducible and constitutive elevations of intracellular hydrogen peroxide (H2O2) have different roles: only the inducible H2O2 production transduces the abscisic acid (ABA) signal leading stomatal closure. However, the involvement of inducible or constitutive NO• productions, if exists, in this process remains unknown. We studied H2O2 and NO• mobilization in guard cells of catalase mutants. Constitutive H2O2 level was higher in the mutants than that in wild type, but constitutive NO• level was not different among lines. Induced NO• and H2O2 levels elicited by ABA showed a high correlation with each other in all lines. Furthermore, NO• levels increased by exogenous H2O2 also showed a high correlation with stomatal aperture size. Our results demonstrate that ABA-induced intracellular H2O2 accumulation triggers NO• production leading stomatal closure. ABBREVIATIONS: ABA: abscisic acid; CAT: catalase; cGMP: cyclic guanosine monophosphate; DAF-2DA: 4,5-diaminofluorescein-2 diacetate; H2DCF-DA: 2',7'-dichlorodihydrofluorescein diacetate; MeJA: methyljasmonate; NOS: nitric oxide synthetase; NR: nitrate reductase; POX: peroxidase; ROS: reactive oxygen species; SNAP: S-nitroso-N-acetyl-DL-penicillamine; SNP: sodium nitroprusside; NOX: NADP(H) oxidase.

MPK9 and MPK12 function in SA-induced stomatal closure in Arabidopsis thaliana.

Salicylic acid (SA) induces stomatal closure sharing several components with abscisic acid (ABA) and methyl jasmonate (MeJA) signaling. We have previously shown that two guard cell-preferential mitogen-activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signaling and MeJA signaling in Arabidopsis thaliana. In this study, we examined whether these two MAPKs are involved in SA-induced stomatal closure using genetic mutants and a pharmacological, MAPKK inhibitor. Salicylic acid induced stomatal closure in mpk9 and mpk12 single mutants but not in mpk9 mpk12 double mutants. The MAPKK inhibitor PD98059 inhibited SA-induced stomatal closure in wild-type plants. Salicylic acid induced extracellular reactive oxygen species (ROS) production, intracellular ROS accumulation, and cytosolic alkalization in the mpk9, mpk12, and mpk9 mpk12 mutants. Moreover, SA-activated S-type anion channels in guard cells of wild-type plants but not in guard cells of mpk9 mpk12 double mutants. These results imply that MPK9 and MPK12 are positive regulators of SA signaling in Arabidopsis guard cells.

Glucosinolate degradation products, isothiocyanates, nitriles, and thiocyanates, induce stomatal closure accompanied by peroxidase-mediated reactive oxygen species production in Arabidopsis thaliana.

Isothiocyanates, nitriles, and thiocyanates are degradation products of glucosinolates in crucifer plants. In this study, we investigated the stomatal response to allyl isothiocyanate (AITC), 3-butenenitrile (3BN), and ethyl thiocyanate (ESCN) in Arabidopsis. AITC, 3BN, and ESCN induced stomatal closure in the wild type and the atrbohD atrbohF mutant. Stomatal closure was inhibited by catalase and salicylhydroxamic acid (SHAM). The degradation products induced extracellular reactive oxygen species (ROS) production in the rosette leaves, and intracellular ROS accumulation, NO production, and cytosolic free calcium concentration ([Ca(2+)]cyt) oscillations in guard cells, which were inhibited by SHAM. These results suggest that glucosinolate degradation products induce stomatal closure accompanied by extracellular ROS production mediated by SHAM-sensitive peroxidases, intracellular ROS accumulation, and [Ca(2+)]cyt oscillation in Arabidopsis.

Endogenous abscisic acid is involved in methyl jasmonate-induced reactive oxygen species and nitric oxide production but not in cytosolic alkalization in Arabidopsis guard cells.

We recently demonstrated that endogenous abscisic acid (ABA) is involved in methyl jasmonate (MeJA)-induced stomatal closure in Arabidopsis thaliana. In this study, we investigated whether endogenous ABA is involved in MeJA-induced reactive oxygen species (ROS) and nitric oxide (NO) production and cytosolic alkalization in guard cells using an ABA-deficient Arabidopsis mutant, aba2-2, and an inhibitor of ABA biosynthesis, fluridon (FLU). The aba2-2 mutation impaired MeJA-induced ROS and NO production. FLU inhibited MeJA-induced ROS production in wild-type guard cells. Pretreatment with 0.1 µM ABA, which does not induce stomatal closure in the wild type, complemented the insensitivity to MeJA of the aba2-2 mutant. However, MeJA induced cytosolic alkalization in both wild-type and aba2-2 guard cells. These results suggest that endogenous ABA is involved in MeJA-induced ROS and NO production but not in MeJA-induced cytosolic alkalization in Arabidopsis guard cells.

Effects of depletion of glutathione on abscisic acid- and methyl jasmonate-induced stomatal closure in Arabidopsis thaliana.

Glutathione (GSH) is involved in abscisic acid (ABA)- and methyl jasmonate (MeJA)-induced stomatal closure in Arabidopsis thaliana. In this study, we examined the effects of GSH-decreasing chemicals, p-nitrobenzyl chloride (PNBC), iodomethane (IDM), and ethacrynic acid (EA), on ABA- and MeJA-induced stomatal closure in Arabidopsis. Treatments with PNBC, IDM, and EA decreased GSH contents in guard cells. Depletion of GSH by PNBC and IDM enhanced ABA- and MeJA-induced stomatal closure and inhibition of light-induced stomatal opening by ABA, whereas EA did not enhance either ABA- and MeJA-induced stomatal closure or inhibition of light-induced stomatal opening by ABA. Depletion of GSH did not significantly increase the production of the reactive oxygen species (ROS), cytosolic alkalization, or cytosolic Ca(2+) oscillation induced by ABA and MeJA. These results indicate that depletion of GSH enhances ABA- and MeJA-induced stomatal closure without affecting ROS production, cytosolic alkalization, or cytosolic Ca(2+) oscillation in guard cells of Arabidopsis.

MAP kinases, MPK9 and MPK12, regulate chitosan-induced stomatal closure.

Chitosan (CHT)-induced stomatal closure was inhibited by an MAPKK inhibitor, PD98059, and was impaired in mpk9 mpk12 but not in mpk9 or mpk12. CHT induced the production of reactive oxygen species, cytosolic alkalization, and cytosolic Ca(2+) oscillation in mpk9 mpk12. These results suggest that MPK9 and MPK12 are involved in CHT-induced stomatal closure.

Lipoprotein status among urban populations in Bangladesh.

OBJECTIVE: Serum lipoprotein is the most important predictor for microvascular diseases, and may be influenced by rapid urbanization. Currently available data are limited, particularly regarding age-specific lipoprotein status in urban Bangladeshi populations. METHODS: Blood lipoprotein levels of 51,353 male and female individuals primarily residing in urban Bangladesh were analyzed. De-identified data (collected between January 2005 and December 2011) were extracted from the Clinical Biochemistry Laboratory Data Archive of International Centre for Diarrheal Disease Research, Bangladesh (icddr,b). For analyses, six age categories were created: (i) <20 years, n = 481; (ii) 20-29 years, n = 1602; (iii) 30-39 years, n = 7272; (iv) 40-49 years, n = 13,582; (v) 50-59 years, n = 15,890; and (vi) 60 years and more, n = 12,526. RESULTS: Mean serum levels of TC, LDL, TG, LDL:HDL and TC:HDL were significantly higher among adults 30-39 years old compared to other age groups, regardless of sex. The proportion of high TC and LDL from 2005 to 2011 among individuals aged 30-39 years old varied widely (p < 0.01 for trend and all pairwise tests). CONCLUSION: 30-39 years old individuals had higher concentration of lipoprotein, which increases microvascular disease risk. Further population-based studies are needed to validate our observations in rural areas of Bangladesh.

Catalases negatively regulate methyl jasmonate signaling in guard cells.

Methyl jasmonate (MeJA)-induced stomatal closure is accompanied by the accumulation of hydrogen peroxide (H2O2) in guard cells. In this study, we investigated the roles of catalases (CATs) in MeJA-induced stomatal closure using cat mutants cat2, cat3-1 and cat1 cat3, and the CAT inhibitor, 3-aminotriazole (AT). When assessed with 2',7'-dichlorodihydrofluorescein, the reduction of catalase activity by means of mutations and the inhibitor accumulated higher basal levels of H2O2 in guard cells whereas they did not affect stomatal aperture in the absence of MeJA. In contrast, the cat mutations and the treatment with AT potentiated MeJA-induced stomatal closure and MeJA-induced H2O2 production. These results indicate that CATs negatively regulate H2O2 accumulation in guard cells and suggest that inducible H2O2 production rather than constitutive elevation modulates stomatal apertures in Arabidopsis.

Cooperative function of PLDδ and PLDα1 in abscisic acid-induced stomatal closure in Arabidopsis.

Phospholipase D (PLD) is involved in responses to abiotic stress and abscisic acid (ABA) signaling. To investigate the roles of two Arabidopsis (Arabidopsis thaliana) PLDs, PLDα1 and PLDδ, in ABA signaling in guard cells, we analyzed ABA responses in guard cells using Arabidopsis wild type, pldα1 and pldδ single mutants, and a pldα1 pldδ double mutant. ABA-induced stomatal closure was suppressed in the pldα1 pldδ double mutant but not in the pld single mutants. The pldα1 and pldδ mutations reduced ABA-induced phosphatidic acid production in epidermal tissues. Expression of either PLDα1 or PLDδ complemented the double mutant stomatal phenotype. ABA-induced stomatal closure in both pldα1 and pldδ single mutants was inhibited by a PLD inhibitor (1-butanol ), suggesting that both PLDα1 and PLDδ function in ABA-induced stomatal closure. During ABA-induced stomatal closure, wild-type guard cells accumulate reactive oxygen species and nitric oxide and undergo cytosolic alkalization, but these changes are reduced in guard cells of the pldα1 pldδ double mutant. Inward-rectifying K(+) channel currents of guard cells were inhibited by ABA in the wild type but not in the pldα1 pldδ double mutant. ABA inhibited stomatal opening in the wild type and the pldδ mutant but not in the pldα1 mutant. In wild-type rosette leaves, ABA significantly increased PLDδ transcript levels but did not change PLDα1 transcript levels. Furthermore, the pldα1 and pldδ mutations mitigated ABA inhibition of seed germination. These results suggest that PLDα1 and PLDδ cooperate in ABA signaling in guard cells but that their functions do not completely overlap.

Methylglyoxal-induced stomatal closure accompanied by peroxidase-mediated ROS production in Arabidopsis.

Methylglyoxal (MG) is an oxygenated short aldehyde and a glycolytic intermediate that accumulates in plants under environmental stresses. Being a reactive α-oxoaldehyde, MG may act as a signaling molecule in plants during stresses. We investigated whether MG induces stomatal closure, reactive oxygen species (ROS) production, and cytosolic free calcium concentration ([Ca²âº](cyt)) to clarify roles of MG in Arabidopsis guard cells. MG induced production of ROS and [Ca²âº](cyt) oscillations, leading to stomatal closure. The MG-induced stomatal closure and ROS production were completely inhibited by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), but were not affected by an NAD(P)H oxidase mutation, atrbohD atrbohF. Furthermore, the MG-elicited [Ca²âº](cyt) oscillations were significantly suppressed by SHAM but not by the atrbohD atrbohF mutation. Neither endogenous abscisic acid nor endogenous methyl jasmonate was involved in MG-induced stomatal closure. These results suggest that intrinsic metabolite MG can induce stomatal closure in Arabidopsis accompanied by extracellular ROS production mediated by SHAM-sensitive peroxidases, intracellular ROS accumulation, and [Ca²âº](cyt) oscillations.

The roles of CATALASE2 in abscisic acid signaling in Arabidopsis guard cells.

We investigated the roles of catalase (CAT) in abscisic acid (ABA)-induced stomatal closure using a cat2 mutant and an inhibitor of CAT, 3-aminotriazole (AT). Constitutive reactive oxygen species (ROS) accumulation due to the CAT2 mutation and AT treatment did not affect stomatal aperture in the absence of ABA, whereas ABA-induced stomatal closure, ROS production, and [Ca(2+)](cyt) oscillation were enhanced.

Negative regulation of abscisic acid-induced stomatal closure by glutathione in Arabidopsis.

We found that glutathione (GSH) is involved in abscisic acid (ABA)-induced stomatal closure. Regulation of ABA signaling by GSH in guard cells was investigated using an Arabidopsis mutant, cad2-1, that is deficient in the first GSH biosynthesis enzyme, γ-glutamylcysteine synthetase, and a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene (CDNB). Glutathione contents in guard cells decreased along with ABA-induced stomatal closure. Decreasing GSH by both the cad2-1 mutation and CDNB treatment enhanced ABA-induced stomatal closure. Glutathione monoethyl ester (GSHmee) restored the GSH level in cad2-1 guard cells and complemented the stomatal phenotype of the mutant. Depletion of GSH did not significantly increase ABA-induced production of reactive oxygen species in guard cells and GSH did not affect either activation of plasma membrane Ca(2+)-permeable channel currents by ABA or oscillation of the cytosolic free Ca(2+) concentration induced by ABA. These results indicate that GSH negatively modulates a signal component other than ROS production and Ca(2+) oscillation in ABA signal pathway of Arabidopsis guard cells.

K252a-sensitive protein kinases but not okadaic acid-sensitive protein phosphatases regulate methyl jasmonate-induced cytosolic Ca2+ oscillation in guard cells of Arabidopsis thaliana.

Methyl jasmonate (MeJA) induces stomatal closure similar to abscisic acid (ABA), and MeJA signaling in guard cells shares some signal components with ABA signaling. As part of this process, MeJA as well as ABA induce the elevation and oscillation of cytosolic free-calcium concentrations ([Ca(2+)](cyt)) in guard cells. While abscisic acid-induced [Ca(2+)](cyt) oscillation has been extensively studied, MeJA-induced [Ca(2+)](cyt) oscillation is less well understood. In this study, we investigated the effects of K252a (a broad-range protein kinase inhibitor) and okadaic acid (OA, a protein phosphatase 1 and 2A inhibitor) on MeJA-induced [Ca(2+)](cyt) oscillation in guard cells of Arabidopsis thaliana ecotype Columbia expressing the Ca(2+) reporter yellow cameleon 3.6. The protein kinase inhibitor K252a abolished MeJA-induced stomatal closure and reduced MeJA-elicited [Ca(2+)](cyt) oscillation. The protein phosphatase inhibitor OA, on the other hand, did not inhibit these processes. These results suggest that MeJA signaling involves activation of K252a-sensitive protein kinases upstream of [Ca(2+)](cyt) oscillation but not activation of an OA-sensitive protein phosphatase in guard cells of A. thaliana ecotype Columbia.

Allyl isothiocyanate (AITC) induces stomatal closure in Arabidopsis.

Isothiocyanates (ITCs) are degradation products of glucosinolates in crucifer plants and have repellent effect on insects, pathogens and herbivores. In this study, we report that exogenously applied allyl isothiocyanate (AITC) induced stomatal closure in Arabidopsis via production of reactive oxygen species (ROS) and nitric oxide (NO), and elevation of cytosolic Ca(2+) . AITC-induced stomatal closures were partially inhibited by an inhibitor of NADPH oxidase and completely inhibited by glutathione monoethyl ester (GSHmee). AITC-induced stomatal closure and ROS production were examined in abscisic acid (ABA) deficient mutant aba2-2 and methyl jasmonate (MeJA)-deficient mutant aos to elucidate involvement of endogenous ABA and MeJA. Genetic evidences have demonstrated that AITC-induced stomatal closure required MeJA priming but not ABA priming. These results raise the possibility that crucifer plants produce ITCs to induce stomatal closure, leading to suppression of water loss and invasion of fungi through stomata.

Mg-chelatase H subunit affects ABA signaling in stomatal guard cells, but is not an ABA receptor in Arabidopsis thaliana.

Mg-chelatase H subunit (CHLH) is a multifunctional protein involved in chlorophyll synthesis, plastid-to-nucleus retrograde signaling, and ABA perception. However, whether CHLH acts as an actual ABA receptor remains controversial. Here we present evidence that CHLH affects ABA signaling in stomatal guard cells but is not itself an ABA receptor. We screened ethyl methanesulfonate-treated Arabidopsis thaliana plants with a focus on stomatal aperture-dependent water loss in detached leaves and isolated a rapid transpiration in detached leaves 1 (rtl1) mutant that we identified as a novel missense mutant of CHLH. The rtl1 and CHLH RNAi plants showed phenotypes in which stomatal movements were insensitive to ABA, while the rtl1 phenotype showed normal sensitivity to ABA with respect to seed germination and root growth. ABA-binding analyses using (3)H-labeled ABA revealed that recombinant CHLH did not bind ABA, but recombinant pyrabactin resistance 1, a reliable ABA receptor used as a control, showed specific binding. Moreover, we found that the rtl1 mutant showed ABA-induced stomatal closure when a high concentration of extracellular Ca(2+) was present and that a knockout mutant of Mg-chelatase I subunit (chli1) showed the same ABA-insensitive phenotype as rtl1. These results suggest that the Mg-chelatase complex as a whole affects the ABA-signaling pathway for stomatal movements.

Involvement of endogenous abscisic acid in methyl jasmonate-induced stomatal closure in Arabidopsis.

In this study, we examined the involvement of endogenous abscisic acid (ABA) in methyl jasmonate (MeJA)-induced stomatal closure using an inhibitor of ABA biosynthesis, fluridon (FLU), and an ABA-deficient Arabidopsis (Arabidopsis thaliana) mutant, aba2-2. We found that pretreatment with FLU inhibited MeJA-induced stomatal closure but not ABA-induced stomatal closure in wild-type plants. The aba2-2 mutation impaired MeJA-induced stomatal closure but not ABA-induced stomatal closure. We also investigated the effects of FLU and the aba2-2 mutation on cytosolic free calcium concentration ([Ca(2+)](cyt)) in guard cells using a Ca(2+)-reporter fluorescent protein, Yellow Cameleon 3.6. In wild-type guard cells, FLU inhibited MeJA-induced [Ca(2+)](cyt) elevation but not ABA-induced [Ca(2+)](cyt) elevation. The aba2-2 mutation did not affect ABA-elicited [Ca(2+)](cyt) elevation but suppressed MeJA-induced [Ca(2+)](cyt) elevation. We also tested the effects of the aba2-2 mutation and FLU on the expression of MeJA-inducible VEGETATIVE STORAGE PROTEIN1 (VSP1). In the aba2-2 mutant, MeJA did not induce VSP1 expression. In wild-type leaves, FLU inhibited MeJA-induced VSP1 expression. Pretreatment with ABA at 0.1 µm, which is not enough concentration to evoke ABA responses in the wild type, rescued the observed phenotypes of the aba2-2 mutant. Finally, we found that in wild-type leaves, MeJA stimulates the expression of 9-CIS-EPOXYCAROTENOID DIOXYGENASE3, which encodes a crucial enzyme in ABA biosynthesis. These results suggest that endogenous ABA could be involved in MeJA signal transduction and lead to stomatal closure in Arabidopsis guard cells.

The Arabidopsis calcium-dependent protein kinase, CPK6, functions as a positive regulator of methyl jasmonate signaling in guard cells.

Previous studies have demonstrated that methyl jasmonate (MeJA) induces stomatal closure dependent on change of cytosolic free calcium concentration in guard cells. However, these molecular mechanisms of intracellular Ca(2+) signal perception remain unknown. Calcium-dependent protein kinases (CDPKs) function as Ca(2+) signal transducers in various plant physiological processes. It has been reported that four Arabidopsis (Arabidopsis thaliana) CDPKs, CPK3, CPK6, CPK4, and CPK11, are involved in abscisic acid signaling in guard cells. It is also known that there is an interaction between MeJA and abscisic acid signaling in guard cells. In this study, we examined the roles of these CDPKs in MeJA signaling in guard cells using Arabidopsis mutants disrupted in the CDPK genes. Disruption of the CPK6 gene impaired MeJA-induced stomatal closure, but disruption of the other CDPK genes did not. Despite the broad expression pattern of CPK6, we did not find other remarkable MeJA-insensitive phenotypes in the cpk6-1 mutant. The whole-cell patch-clamp analysis revealed that MeJA activation of nonselective Ca(2+)-permeable cation channels is impaired in the cpk6-1 mutant. Consistent with this result, MeJA-induced transient cytosolic free calcium concentration increments were reduced in the cpk6-1 mutant. MeJA failed to activate slow-type anion channels in the cpk6-1 guard cells. Production of early signal components, reactive oxygen species and nitric oxide, in guard cells was elicited by MeJA in the cpk6-1 mutant as in the wild type. These results provide genetic evidence that CPK6 has a different role from CPK3 and functions as a positive regulator of MeJA signaling in Arabidopsis guard cells.

Involvement of extracellular oxidative burst in salicylic acid-induced stomatal closure in Arabidopsis.

Salicylic acid (SA), a ubiquitous phenolic phytohormone, is involved in many plant physiological processes including stomatal movement. We analysed SA-induced stomatal closure, production of reactive oxygen species (ROS) and nitric oxide (NO), cytosolic calcium ion ([Ca²+](cyt)) oscillations and inward-rectifying potassium (K+(in)) channel activity in Arabidopsis. SA-induced stomatal closure was inhibited by pre-treatment with catalase (CAT) and superoxide dismutase (SOD), suggesting the involvement of extracellular ROS. A peroxidase inhibitor, SHAM (salicylhydroxamic acid) completely abolished SA-induced stomatal closure whereas neither an inhibitor of NADPH oxidase (DPI) nor atrbohD atrbohF mutation impairs SA-induced stomatal closures. 3,3'-Diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) stainings demonstrated that SA induced H2O2 and O2⁻ production. Guard cell ROS accumulation was significantly increased by SA, but that ROS was suppressed by exogenous CAT, SOD and SHAM. NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) suppressed the SA-induced stomatal closure but did not suppress guard cell ROS accumulation whereas SHAM suppressed SA-induced NO production. SA failed to induce [Ca²+](cyt) oscillations in guard cells whereas K+(in) channel activity was suppressed by SA. These results indicate that SA induces stomatal closure accompanied with extracellular ROS production mediated by SHAM-sensitive peroxidase, intracellular ROS accumulation and K+(in) channel inactivation.

The involvement of intracellular glutathione in methyl jasmonate signaling in Arabidopsis guard cells.

We examined the involvement of intracellular glutathione (GSH) in methyl jasmonate (MeJA) signaling. The chlorina1-1 (ch1-1) mutation decreased GSH in guard cells and narrowed the stomatal aperture. GSH monoethyl ester increased intracellular GSH, diminishing this phenotype. GSH did not affect MeJA-induced reactive oxygen species production or cytosolic Ca(2+) oscillation, suggesting that GSH modulates MeJA signaling downstream of production and oscillation.

Yeast elicitor-induced stomatal closure and peroxidase-mediated ROS production in Arabidopsis.

Yeast elicitor (YEL) induces stomatal closure. We investigated reactive oxygen species (ROS) production, nitric oxide (NO) production and [Ca(2+)](cyt) oscillations to clarify YEL signaling in Arabidopsis guard cells. YEL induced ROS accumulation in guard cells. A peroxidase inhibitor [salicylhydroxamic acid (SHAM)] inhibited the stomatal closure and the ROS accumulation, but neither the atrbohD atrbohF mutation nor an NADPH oxidase inhibitor [diphenylene iodonium chloride (DPI)] had any effect. An NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO)] inhibited the YEL-induced stomatal closure and SHAM abolished NO production. YEL-elicited [Ca(2+)](cyt) oscillations were inhibited by SHAM but not by the atrbohD atrbohF mutation. These results indicate that YEL induces stomatal closure accompanied by ROS production mediated by peroxidases and NO production.