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.

Inhibition of ABA-induced stomatal closure by fusicoccin is associated with cytosolic acidification-mediated hydrogen peroxide removal.

BACKGROUND: Fusicoccin (FC), a fungal phytotoxin produced by Fusicoccum amygdale, causes the inhibition of ABA-induced stomatal closure. The mechanism of inhibition is remaining unclear. We analyzed the role of hydrogen peroxide (H2O2) and relationship between H2O2 removal and cytosolic pH changes during inhibition of ABA-induced stomatal closure by FC. RESULTS: According to the results, ABA treatment induced H2O2 production and stomatal closure, but FC inhibited the effects of ABA on these two parameters. Treatment with catalase (CAT) and NADPH oxidase inhibitor diphenylene iodonium (DPI) mimicked the effect of FC. These data suggest that inhibition of ABA effect by FC is related to the decrease of H2O2 levels in guard cells. Furthermore, similar to CAT, FC not only suppressed stomatal closure and H2O2 levels in guard cells treated with exogenous H2O2, but also reopened the stomata which had been closed by ABA and reduced the level of H2O2 that had been produced by ABA, indicating that FC causes H2O2 removal in guard cells. The butyric acid treatment simulated the effects of FC on the stomatal aperture and H2O2 levels in guard cells treated with exogenous H2O2 and had been closed by ABA, and both FC and butyric acid reduced cytosolic pH in guard cells of stomata treated with H2O2 and had been closed by ABA, which demonstrate that cytosolic acidification mediates FC-induced H2O2 removal. CONCLUSION: These results suggest that FC causes cytosolic acidification in guard cells, then induces H2O2 removal and reduces H2O2 levels in guard cells, finally inhibits stomatal closure induced by ABA.

Actin microfilaments and vacuoles are downstream targets of H2O2 signalling pathways in hyperosmotic stress-induced stomatal closure.

Changes in osmotic pressure can induce stomatal closure to reduce transpirational water loss from plants. In the present work, we investigated the mechanism underlying the perception and transduction of extracellular changes in osmotic pressure in Vicia faba L. guard cells. Using an epidermal strip bioassay and laser-scanning confocal microscopy, we provide evidence that hyperosmotic stress treatment led to stomatal closure and the rapid promotion of hydrogen peroxide (H2O2) production in V. faba guard cells. The effects were largely reduced by H2O2 scavengers ASA, CAT, NADPH oxidase inhibitor DPI and cell wall peroxidase inhibitor SHAM. These results indicate that hyperosmotic stress induces stomatal closure by promoting H2O2 production. Cytochalasin B (CB), latrunculin B (Lat B) and jasplakinolide (JK) inhibited stomatal closure induced by hyperosmotic stress but didn't prevent the increase of endogenous H2O2 levels, suggesting that microfilaments reorganisation participates in stomatal closure induced by hyperosmotic stress, and may act downstream of H2O2 signalling processes. In addition, we observed splitting of big vacuoles into many small vacuoles in response to hyperosmotic stress and H2O2 treatment, and CB inhibited these changes of vacuoles; stomatal closure was also inhibited. Taken together these results indicate that the stomatal closure in response to hyperosmotic stress may initiate H2O2 generation, and that reorganisation of microfilaments and the changing of vacuoles occurs downstream of H2O2 signalling processes.

Ethylene inhibits darkness-induced stomatal closure by scavenging nitric oxide in guard cells of Vicia faba.

The plant hormone ethylene regulates many aspects of plant growth and development. Despite the well-known relationship between ethylene and stress signalling, the involvement of ethylene in regulating stomatal movement is not completely explored. Here, the role and association between nitric oxide (NO) reduction and the inhibition of darkness-induced stomatal closure by ethylene was studied. Physiological data are provided that both ethylene-releasing compound 2-chloroethylene phosphonic acid (ethephon, ETH) and 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, reduced the levels of NO in Vicia faba L. guard cells, and then induced stomatal opening in darkness. In addition, ACC and ETH not only reduced NO levels in guard cells caused by exogenous NO (derived from sodium nitroprusside, SNP) in light, but also abolished NO that had been generated during a dark period and promoted stomatal opening. Interestingly, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and hemoglobin (Hb), NO scavenger and the potent scavenger of NO/carbon monoxide (CO), respectively, also reduced NO levels by SNP and darkness. However, the above-mentioned effects of ACC and ETH were dissimilar to that of nitric oxide synthase (enzyme commission 1.14.13.39) inhibitor NG-nitro-L-Arg-methyl ester (L-NAME), which could neither reduce NO levels by SNP nor abolish NO that had been generated in the dark. Thus, it is concluded that ethylene reduces the levels of NO in V. faba guard cells via a pattern of NO scavenging, then induces stomatal opening in the dark.

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

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

Nitric oxide, actin reorganization and vacuoles change are involved in PEG 6000-induced stomatal closure in Vicia faba.

Water deficit and the resulting osmotic stress affect stomatal movement. There are two types of signals, hydraulic and chemical signals, involving in the regulation of stomatal behavior responses to osmotic stress. Compared with the chemical signals, little has been known about the hydraulic signals and the corresponding signal transduction network and regulatory mechanisms. Here, using an epidermal-strip bioassay and laser-scanning confocal microscopy, we provide evidence that nitric oxide (NO) generation in Vicia faba guard cells can be induced by hydraulic signals. We used polyethylene glycol (PEG) 600 to simulate hypertonic conditions. This hydraulic signal led to stomatal closure and rapid promotion of NO production in guard cells. The effects were decreased by NO scavenger 2-(4-carboxyphenyl)-4,4,5, 5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) and NO synthase (Enzyme Commission 1.14.13.39) inhibitor N(G)-nitro-L-Arg-methyl ester (L-NAME). These results indicate that PEG 6000 induces stomatal closure by promoting NO production. Cytochalasin B (CB) inhibited stomatal closure induced by PEG 6000 but did not prevent the increase of endogenous NO levels, indicating that microfilaments polymerization participate in stomatal closure induced by PEG 6000, and may act downstream of NO signaling. In addition, big vacuoles split into many small vacuoles were observed in response to PEG 6000 and sodium nitroprusside (SNP) treatment, and CB inhibited these changes of vacuoles, the stomatal closure was also been inhibited. Collectively, these results suggest that the stomatal closure induced by PEG 6000 may be intimately associated with NO levels, reorganization of actin filaments and the changes of vacuoles, showing a crude outline of guard-cells signaling process in response to hydraulic signals.

Carbon monoxide-induced stomatal closure involves generation of hydrogen peroxide in Vicia faba guard cells.

Here the regulatory role of CO during stomatal movement in Vicia faba L. was surveyed. Results indicated that, like hydrogen peroxide (H(2)O(2)), CO donor Hematin induced stomatal closure in dose- and time-dependent manners. These responses were also proven by the addition of gaseous CO aqueous solution with different concentrations, showing the first time that CO and H(2)O(2) exhibit the similar regulation role in the stomatal movement. Moreover, our data showed that ascorbic acid (ASA, an important reducing substrate for H(2)O(2) removal) and diphenylene iodonium (DPI, an inhibitor of the H(2)O(2)-generating enzyme NADPH oxidase) not only reversed stomatal closure by CO, but also suppressed the H(2)O(2) fluorescence induced by CO, implying that CO induced-stomatal closure probably involves H(2)O(2) signal. Additionally, the CO/NO scavenger hemoglobin (Hb) and CO specific synthetic inhibitor ZnPPIX, ASA and DPI reversed the darkness-induced stomatal closure and H(2)O(2) fluorescence. These results show that, perhaps like H(2)O(2), the levels of CO in guard cells of V. faba are higher in the dark than in light, HO-1 and NADPH oxidase are the enzyme systems responsible for generating endogenous CO and H(2)O(2) in darkness respectively, and that CO is involved in darkness-induced H(2)O(2) synthesis in V. faba guard cells.

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

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

The involvement of nitric oxide in ultraviolet-B-inhibited pollen germination and tube growth of Paulownia tomentosa in vitro.

The role of nitric oxide (NO) in the ultraviolet-B radiation (UV-B)-induced reduction of in vitro pollen germination and tube growth of Paulownia tomentosa Steud. was studied. Results showed that exposure of the pollen to 0.4 and 0.8 W m(-2) UV-B radiation for 2 h resulted in not only the reduction of pollen germination and tube growth but also the enhancement of NO synthase (NOS, EC 1.14.13.39) activity and NO production in pollen grain and tube. Also, exogenous NO donors sodium nitroprusside and S-nitrosoglutathione inhibited both pollen germination and tube growth in a dose-dependence manner. NOS inhibitor N(G)-nitro-l-Arg-methyl eater (l-NAME) and NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) not only largely prevented the NO generation but also partly reversed the UV-B-inhibited pollen germination and tube growth. These results indicate that UV-B radiation inhibits pollen germination and tube growth partly via promoting NO production in pollen grain and tube by a NOS-like enzyme. Additionally, a guanylyl cyclase inhibitor 6-anilino-5,8-quinolinequinone (LY-83583) prevented both the UV-B- and NO donors-inhibited pollen germination and tube growth, suggesting that the NO function is mediated by cyclic guanosine 5'-monophosphate. However, the effects of c-PTIO, l-NAME and LY-83583 on the UV-B-inhibited pollen germination and tube growth were only partial, suggesting that there are NO-independent pathways in UV-B signal networks.

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.

The role and the interrelationship of hydrogen peroxide and nitric oxide in the UV-B-induced stomatal closure in broad bean.

Previous studies have showed that UV-B can stimulate closure as well as opening of stomata. However, the mechanism of this complex effect of UV-B is not clear. The purpose of this paper is to investigate the role and the interrelationship of H2O2 and NO in UV-B-induced stomatal closure in broad bean (Vicia faba L.). By epidermal strip bioassay and laser-scanning confocal microscopy, we observed that UV-B-induced stomatal closure could be largely prevented not only by NO scavenger c-PTIO or NO synthase (NOS) inhibitor l-NAME, but also by ascorbic acid (ASC, an important reducing substrate for H2O2 removal) or catalase (CAT, the H2O2 scavenger), and that UV-B-induced NO and H2O2 production in guard cells preceded UV-B-induced stomatal closure. These results indicate that UV-B radiation induces stomatal closure by promoting NO and H2O2 production. In addition, c-PTIO, l-NAME, ASC and CAT treatments could effectively inhibit not only UV-B-induced NO production, but also UV-B-induced H2O2 production. Exogenous H2O2-induced NO production and stomatal closure were partly abolished by c-PTIO and l-NAME. Similarly, exogenous NO donor sodium nitroprusside-induced H2O2 production and stomatal closure were also partly reversed by ASC and CAT. These results show a causal and interdependent relationship between NO and H2O2 during UV-B-regulated stomatal movement. Furthermore, the l-NAME data also indicate that the NO in guard cells of Vicia faba is probably produced by a NOS-like enzyme.

Reduction of Rubisco amount by UV-B radiation is related to increased H2O2 content in leaves of mung bean seedlings.

To understand the role of H(2)O(2) in the damaging effect of UV-B radiation on ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) content, the time courses of changes in Rubisco and H(2)O(2) contents under UV-B treatment were measured in the primary leaves of two mung bean cultivars (Phaseolus raditus L. cv. Zhonglv-1 and Qindou-20). A 0.4 W/m(2) UV-B treatment caused a drop of the former to about 1/3 and a 30%-40% rise in H(2)O(2) content in a week in both cultivars but with Qindou-20 showing stronger responses. Such higher rate of increase in proteolytic activity under UV-B treatment implies that UV-B lowers Rubisco by activating proteolytic enzymes which hydrolizes Rubisco. Scavenging of H(2)O(2) by exogenously added ascorbic acid (AsA) significantly inhibited the UV-B-induced decrease in Rubisco amount and increases in H(2)O(2) level and proteolytic activity. It is shown that H(2)O(2) participates in the UV-B-induced reduction of Rubisco amount. It is suggested that H(2)O(2) stimulates the degradation of Rubisco by activation of proteolytic systems.

[Stomatal and nonstomatal limitations of photosynthesis in mung bean leaves under the combination of enhanced UV-B radiation and NaCl stress].

Stomatal and nonstomatal limitations of photosynthesis in mung bean (Phaseolus radiatus L.) leaves under the combination of 0.35 W/m(2) UV-B radiation and 0.4% NaCl stress were studied. Separated or combined treatments of enhanced UV-B radiation and NaCl stress all resulted in a decrease in net photosynthetic rate, stomatal conductance, photosynthetic ability, efficiency of CO(2) carboxylation and Rubisco content, and the extent of those decreases enhanced obviously with the increase of treatment days. Intercellular CO(2)concentration tended to decrease in short period, followed by a gradual increase and to be higher than the control on the seventh day after treatment under NaCl stress alone, while a tendency of gradual increase was observed under enhanced UV-B radiation alone or two stresses combined and the concentration was higher than the control after three day of treatments. By contrast, the tendency of stomatal limitation value was just reverse to the intercellular CO(2) concentration, but the stomatal limitation value was always higher than control under all stress conditions (did not include that on the fifth day under the combined stresses). As compared with an individual stress the effect of combined stress on the above parameters was more serious. These results indicate that under all stress conditions the inhibition of photosynthesis in mung bean leaves is the results of both stomatal and nonstomatal limitations. And the stomatal limitation is dominant in short period, nonstomatal limitation becomes the dominant one in longer period. The decrease in Rubisco content leads to nonstomatal limitation of photosynthesis under all stresses.