H2O2 In Guard Cells Research Articles

MdBAM17, a novel member of the β-amylase gene family, positively regulates starch degradation in ALA-induced stomatal opening in apple (Malus × domestica)

5-Aminolevulinic acid (ALA) is a novel plant growth regulator that has shown outstanding capability to promote stomatal opening. Starch degradation, catalyzed by β-amylase (EC3.2.1.2, BAM), plays an important role in stomatal opening. However, whether the starch breakdown is involved in ALA-regulating stomatal movement is unclear. In the current study, we found that exogenous ALA effectively stimulated the starch breakdown in guard cells, increased β-amylase activity and promoted stomatal opening in leaves of apple (Malus × domestica). Based on genome-wide identification, we identified a total of 119 members of BAM gene family in ten commonly Rosaceae crops. Analyses of gene structure, motif identification, and gene pair collinearity revealed relative conservation among members within the same group or subgroup. Among these genes, MdBAM17 and other 12 genes were identified as the orthologous genes of AtBAM1, which is responsible for starch degradation to modulate the stomatal movement in Arabidopsis. RT-qPCR analysis revealed a positive correlation between the expressions of MdBAM17 and stomatal aperture, as well as β-amylase activity, whereas a negative correlation was observed with the starch content. Subcellular localization analysis confirmed that MdBAM17 is a chloroplast protein, consistent with the AtBAM1. MdBAM17 was mainly expressed in guard cells and responsive to exogenous ALA. Overexpressing MdBAM17 increased β-amylase activity and promoted starch breakdown, leading to stomatal opening, which was further strengthened by ALA. RNA-interfering MdBAM17 decreased β-amylase activity, resulting in starch accumulation, and impairing the stomatal opening by ALA. However, modulation of MdBAM17 expression did not affect the levels of flavonols and H2O2 in guard cells, suggesting that MdBAM17-promoted starch degradation may function at downstream of ROS signaling in the ALA-regulated stomatal opening. Our findings provide new insights into the mechanisms of ALA-regulated stomatal movement.

Phospholipase Dδ and H2S increase the production of NADPH oxidase-dependent H2O2 to respond to osmotic stress-induced stomatal closure in Arabidopsis thaliana

Osmotic stress is one of the main stresses that seriously affects the survival of plants, destroying normal cell activities, and potentially leading to plant death. Phospholipase D (PLD), a major lipid hydrolase, hydrolyzes membrane phospholipids to produce phosphatidic acid (PA) and responds to many abiotic stresses. Hydrogen sulfide (H2S) emerges as the third gaseous signaling molecule involved in the complex network of signaling events. Hydrogen peroxide (H2O2) plays a crucial role as a signaling molecule in plant development and growth, and responds to various abiotic and biotic stresses. In this study, the functions and the relationship of PLDδ, H2S, and H2O2 in osmotic stress-induced stomatal closure were explored. By using the seedlings of ecotype (WT), PLDδ-deficient mutant (pldδ), l-cysteine desulfhydrase (LCD)-deficient mutant (lcd), and pldδlcd double mutant, atrbohD, and atrbohF mutant as materials, and the stomatal aperture were analyzed. The relative water loss of pldδ, lcd, and pldδlcd was higher than that of WT. Exogenous PA and NaHS could partially alleviate the leaf wilting and yellowing phenotypes of pldδ, lcd, and pldδlcd under osmotic stress, but the mutants could not be restored to the same phenotype as WT. The fluorescence intensity of H2O2 in guard cells of pldδ, lcd, and pldδlcd was lower than that of WT, indicating that PLDδ and LCD were involved in the production of H2O2 in guard cells. Exogenous application of H2O2 to WT, pldδ, lcd, and pldδlcd significantly induced stomatal closure under osmotic stress. Exogenous NaHS induced stomatal closure of WT, but could not induce stomatal closure of atrbohD and atrbohF under osmotic stress. These results suggest that the accumulation of H2O2 was essential to induce stomatal closure under osmotic stress, and PLDδ and LCD acted upstream of H2O2.

Polyamine Oxidation Is Indispensable for Wheat (Triticum aestivum L.) Oxidative Response and Necrotic Reactions during Leaf Rust (Puccinia triticina Eriks.) Infection.

Hydrogen peroxide is a signal and effector molecule in the plant response to pathogen infection. Wheat resistance to Puccinia triticina Eriks. is associated with necrosis triggered by oxidative burst. We investigated which enzyme system dominated in host oxidative reaction to P. triticina infection. The susceptible Thatcher cultivar and isogenic lines with defined resistance genes were inoculated with P. triticina spores. Using diamine oxidase (DAO) and polyamine oxidase (PAO) inhibitors, accumulation of H2O2 was analyzed in the infection sites. Both enzymes participated in the oxidative burst during compatible and incompatible interactions. Accumulation of H2O2 in guard cells, i.e., the first phase of the response, depended on DAO and the role of PAO was negligible. During the second phase, the patterns of H2O2 accumulation in the infection sites were more complex. Accumulation of H2O2 during compatible interaction (Thatcher and TcLr34 line) moderately depended on DAO and the reaction of TcLr34 was stronger than that of Thatcher. Accumulation of H2O2 during incompatible interaction of moderately resistant plants (TcLr24, TcLr25 and TcLr29) was DAO-dependent in TcLr29, while the changes in the remaining lines were not statistically significant. A strong oxidative burst in resistant plants (TcLr9, TcLr19, TcLr26) was associated with both enzymes’ activities in TcLr9 and only with DAO in TcLr19 and TcLr26. The results are discussed in relation to other host oxidative systems, necrosis, and resistance level.

PP2A and microtubules function in 5‐aminolevulinic acid‐mediated H2O2 signaling in Arabidopsis guard cells

5-aminolevulinic acid (ALA), a plant growth regulator with great application potential in agriculture and horticulture, induces stomatal opening and inhibits stomatal closure by decreasing guard cell H2 O2 . However, the mechanisms behind ALA-decreased H2 O2 in guard cells are not fully understood. Here, using type 2A protein phosphatase (PP2A) inhibitors, microtubule-stabilizing/disrupting drugs and green fluorescent protein-tagged α-tubulin 6 transgenic Arabidopsis (GFP-TUA6), we find that PP2A and cortical microtubules (MTs) are involved in ALA-regulated stomatal movement. Then, we analyze stomatal responses of Arabidopsis overexpressing C2 catalytic subunit of PP2A (PP2A-C2) and pp2a-c2 mutant to ALA and abscisic acid (ABA) under both light and dark conditions, and show that PP2A-C2 participates in ALA-induced stomatal movement. Furthermore, using pharmacological methods and confocal studies, we reveal that PP2A and MTs function upstream and downstream, respectively, of H2 O2 in guard cell signaling. Finally, we demonstrate the role of H2 O2 -mediated microtubule arrangement in ALA inhibiting ABA-induced stomatal closure. Our findings indicate that MTs regulated by PP2A-mediated H2 O2 decreasing play an important role in ALA guard cell signaling, revealing new insights into stomatal movement regulation.

Hydrogen sulfide may function downstream of hydrogen peroxide in CdCl2-induced stomatal closure in Vigna radiata L.

Pharmacological, spectrophotographic and laser scanning confocal microscopy (LSCM) approaches were used to investigate the roles of hydrogen sulfide (H2S) and hydrogen peroxide (H2O2) played in signaling transduction of stomatal closure induced by CdCl2 in Vigna radiata L. In the present study, CdCl2 could induce stomatal closure in a dose- and time-dependent manner, which was inhibited by H2S modulators hypotaurine (HT), aminooxy acetic acid (AOA), hydroxylamine (NH2OH), potassium pyruvate (C3H3KO3), ammonia (NH3), H2O2 modulators ascorbic acid (ASA), catalase (CAT), diphenylene iodonium (DPI) and salicylhydroxamic acid (SHAM). In addition, both the content of H2S, activity of L-/D-cysteine desulfhydrase (L-/D-CDes and pyridoxalphosphate-dependent enzyme) in leaves of V. radiata, and the level of H2O2 in guard cells significantly increased under CdCl2 stress, which were inhibited by H2S and H2O2 modulators, respectively. H2O2 modulators could suppress CdCl2-induced increase of H2S levels, L-/D-CDes activity and stomatal closure in the leaves of V. radiata. However, H2S modulators had no effect on the accumulation of CdCl2-induced H2O2 in guard cells of V. radiata. From these data, it can be deduced that both H2S and H2O2 can mediate CdCl2-induced stomatal closure in V. radiata L., and H2S acts downstream than H2O2 in such process.

NADK2 positively modulates abscisic acid-induced stomatal closure by affecting accumulation of H2O2, Ca2+ and nitric oxide in Arabidopsis guard cells

NAD kinase2 (NADK2) plays key roles in chloroplastic NADP biosynthesis, stress adaptation and modulation of cellular metabolisms in Arabidopsis. However, it is unknown whether and how NADK2 affects abscisic acid (ABA)-mediated stomatal movement. Here, we detected that null mutant nadk2 was more sensitive to drought stress than WT, and NADK2 gene was active in guard cells. Furthermore, NADK2 mutation impaired ABA-induced stomatal closure and ABA inhibition of light-promoted stomatal opening. NADK2 disruption also impaired ABA-stimulated accumulation of H2O2, Ca2+ and nitric oxide (NO) in guard cells, but did not affect the stomatal closure evoked by exogenous H2O2, Ca2+ or NO. Expression analysis revealed that ABA-promoted increases in transcripts of AtrbohD, AtrbohF and NIA1 were markedly arrested in guard cells of nadk2 compared with those of WT. Besides, genetic evidence indicated that NADK2 acted synergistically with OST1 and ABI1 during ABA-induced stomatal closure. Together, these results suggest that NADK2 is an essential positive regulator, and functions upstream of H2O2 in guard cell ABA signaling. It stimulates stomatal closure mainly through increasing the generation of H2O2, Ca2+ and NO in guard cells in Arabidopsis.

ALA-Induced Flavonols Accumulation in Guard Cells Is Involved in Scavenging H2O2 and Inhibiting Stomatal Closure in Arabidopsis Cotyledons.

5-aminolevulinic acid (ALA), a new plant growth regulator, can inhibit stomatal closure by reducing H2O2 accumulation in guard cells. Flavonols are a main kind of flavonoids and have been proposed as H2O2 scavengers in guard cells. 5-aminolevulinic acid can significantly improve flavonoids accumulation in plants. However, whether ALA increases flavonols content in guard cells and the role of flavonols in ALA-regulated stomatal movement remains unclear. In this study, we first demonstrated that ALA pretreatment inhibited ABA-induced stomatal closure by reducing H2O2 accumulation in guard cells of Arabidopsis seedlings. This result confirms the inhibitory effect of ALA on stomatal closure and the important role of decreased H2O2 accumulation in this process. We also found that ALA significantly improved flavonols accumulation in guard cells using a flavonol-specific dye. Furthermore, using exogenous quercetin and kaempferol, two major components of flavonols in Arabidopsis leaves, we showed that flavonols accumulation inhibited ABA-induced stomatal movement by suppressing H2O2 in guard cells. Finally, we showed that the inhibitory effect of ALA on ABA-induced stomatal closure was largely impaired in flavonoid-deficient transparent testa4 (tt4) mutant. In addition, exogenous flavonols recovered stomatal responses of tt4 to the wild-type levels. Taken together, we conclude that ALA-induced flavonol accumulation in guard cells is partially involved in the inhibitory effect of ALA on ABA-induced H2O2 accumulation and stomatal closure. Our data provide direct evidence that ALA can regulate stomatal movement by improving flavonols accumulation, revealing new insights into guard cell signaling.

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.

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.

Nitric oxide, stomatal closure, and abiotic stress

Various data indicate that nitric oxide (NO) is an endogenous signal in plants that mediates responses to several stimuli. Experimental evidence in support of such signalling roles for NO has been obtained via the application of NO, usually in the form of NO donors, via the measurement of endogenous NO, and through the manipulation of endogenous NO content by chemical and genetic means. Stomatal closure, initiated by abscisic acid (ABA), is effected through a complex symphony of intracellular signalling in which NO appears to be one component. Exogenous NO induces stomatal closure, ABA triggers NO generation, removal of NO by scavengers inhibits stomatal closure in response to ABA, and ABA-induced stomatal closure is reduced in mutants that are impaired in NO generation. The data indicate that ABA-induced guard cell NO generation requires both nitric oxide synthase-like activity 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 events are required for ABA-induced stomatal closure. ABA also stimulates the generation of H2O2 in guard cells, and pharmacological and genetic data demonstrate that NO accumulation in these cells is dependent on such production. Recent data have extended this model to maize mesophyll cells where the induction of antioxidant defences by water stress and ABA required the generation of H2O2 and NO and the activation of a MAPK. Published data suggest that drought and salinity induce NO generation which activates cellular processes that afford some protection against the oxidative stress associated with these conditions. Exogenous NO can also protect cells against oxidative stress. Thus, the data suggest an emerging model of stress responses in which ABA has several ameliorative functions. These include the rapid induction of stomatal closure to reduce transpirational water loss and the activation of antioxidant defences to combat oxidative stress. These are two processes that both involve NO as a key signalling intermediate.

Cytokinin- and auxin-induced stomatal opening involves a decrease in levels of hydrogen peroxide in guard cells of Vicia faba.

Previous studies have shown that cytokinins and auxins can induce the opening of stomata. However, the mechanism of stomatal opening caused by cytokinins and auxins remains unclear. The purpose of this paper is to investigate the relationship between hydrogen peroxide (H2O2) levels in guard cells and stomatal opening induced by cytokinins and auxins in Vicia faba. By means of stomatal bioassay and laser-scanning confocal microscopy, we provide evidence that cytokinins and auxins reduced the levels of H2O2 in guard cells and induced stomatal opening in darkness. Additionally, cytokinins not only reduced exogenous H2O2 levels in guard cells caused by exposure to light, but also abolished H2O2 that had been generated during a dark period, and promoted stomatal opening, as did ascorbic acid (ASA, an important reducing substrate for H2O2 removal). However, unlike cytokinins, auxins did not reduce exogenous H2O2, did not abolish H2O2 that had been generated in the dark, and therefore did not promote reopening of stoma induced to close in the dark. The above-mentioned effects of auxins were similar to that of diphenylene iodonium (DPI, an inhibitor of the H2O2-generating enzyme NADPH oxidase). Taken together our results indicate that cytokinins probably reduce the levels of H2O2 in guard cells by scavenging, whereas auxins limit H2O2 levels through restraining H2O2 generation, inducing stomatal opening in darkness.

Extracellular calmodulin-induced stomatal closure is mediated by heterotrimeric G protein and H2O2.

Extracellular calmodulin (ExtCaM) exerts multiple functions in animals and plants, but the mode of ExtCaM action is not well understood. In this paper, we provide evidence that ExtCaM stimulates a cascade of intracellular signaling events to regulate stomatal movement. Analysis of the changes of cytosolic free Ca2+ ([Ca2+]cyt) and H2O2 in Vicia faba guard cells combined with epidermal strip bioassay suggests that ExtCaM induces an increase in both H2O2 levels and [Ca2+]cyt, leading to a reduction in stomatal aperture. Pharmacological studies implicate heterotrimeric G protein in transmitting the ExtCaM signal, acting upstream of [Ca2+]cyt elevation, and generating H2O2 in guard cell responses. To further test the role of heterotrimeric G protein in ExtCaM signaling in stomatal closure, we checked guard cell responses in the Arabidopsis (Arabidopsis thaliana) Galpha-subunit-null gpa1 mutants and cGalpha overexpression lines. We found that gpa1 mutants were insensitive to ExtCaM stimulation of stomatal closure, whereas cGalpha overexpression enhanced the guard cell response to ExtCaM. Furthermore, gpa1 mutants are impaired in ExtCaM induction of H2O2 generation in guard cells. Taken together, our results strongly suggest that ExtCaM activates an intracellular signaling pathway involving activation of a heterotrimeric G protein, H2O2 generation, and changes in [Ca2+]cyt in the regulation of stomatal movements.

Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells.

Drought is a major threat to agricultural production. Plants synthesize the hormone abscisic acid (ABA) in response to drought, triggering a signalling cascade in guard cells that results in stomatal closure, thus reducing water loss. ABA triggers an increase in cytosolic calcium in guard cells ([Ca2+]cyt) that has been proposed to include Ca2+ influx across the plasma membrane. However, direct recordings of Ca2+ currents have been limited and the upstream activation mechanisms of plasma membrane Ca2+ channels remain unknown. Here we report activation of Ca2+-permeable channels in the plasma membrane of Arabidopsis guard cells by hydrogen peroxide. The H2O2-activated Ca2+ channels mediate both influx of Ca2+ in protoplasts and increases in [Ca2+]cyt in intact guard cells. ABA induces the production of H2O2 in guard cells. If H2O2 production is blocked, ABA-induced closure of stomata is inhibited. Moreover, activation of Ca2+ channels by H2O2 and ABA- and H2O2-induced stomatal closing are disrupted in the recessive ABA-insensitive mutant gca2. These data indicate that ABA-induced H2O2 production and the H2O2-activated Ca2+ channels are important mechanisms for ABA-induced stomatal closing.