Dark-induced Stomatal Closure Research Articles

Inhibition of darkness-induced stomatal closure by ethylene involves a removal of hydrogen peroxide from guard cells of Vicia faba

Ethylene regulates many aspects of plant growth and development; however, its effect on the behavior of the stomata is still largely obscure. Here, the association between ethylene inhibition of darkness-induced stomatal closure and hydrogen peroxide (H2O2) levels in Vicia faba guard cells was studied. Like ascorbic acid (ASA), the most important reducing substrate for H2O2 removal, catalase (CAT), one of H2O2-scavenging enzymes, and diphenylene iodonium (DPI), an inhibitor of the H2O2-generating enzyme NADPH-oxidase, both 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 darkness and to reduce H2O2 levels in guard cells, indicating that ethylene-caused inhibition of darkness-induced stomatal closure involves reduction in the H2O2 level in guard cells. Additionally, similar to ASA and CAT, ACC/ETH not only suppressed H2O2-induced stomatal closure and H2O2 level in guard cells treated with exogenous H2O2 in the light, but also reopened the stomata, which had been closed by darkness, and reduced H2O2 level that had been generated by darkness, showing that ethylene causes H2O2 removal from guard cells. However, the above-mentioned effect of ACC/ETH was dissimilar from that of DPI, which not only was incapable to reduce H2O2 level induced by exogenous H2O2 but also could not abolish H2O2 that had been generated by darkness. Thus, we suggest that ethylene probably induces H2O2 removal and reduces H2O2 level in guard cells and finally inhibits stomatal closure induced by darkness. Furthermore, the mechanism of H2O2 removal caused by ethylene was also discussed.

Expression and manipulation of PHOSPHOENOLPYRUVATE CARBOXYKINASE 1 identifies a role for malate metabolism in stomatal closure

Malate, along with potassium and chloride ions, is an important solute for maintaining turgor pressure during stomatal opening. Although malate is exported from guard cells during stomatal closure, there is controversy as to whether malate is also metabolised. We provide evidence that phosphoenolpyruvate carboxykinase (PEPCK), an enzyme involved in malate metabolism and gluconeogenesis, is necessary for full stomatal closure in the dark. Analysis of the Arabidopsis PCK1 gene promoter indicated that this PEPCK isoform is specifically expressed in guard cells and trichomes of the leaf. Spatially distinct promoter elements were found to be required for post-germinative, vascular expression and guard cell/trichome expression of PCK1. We show that pck1 mutant plants have reduced drought tolerance, and show increased stomatal conductance and wider stomatal apertures compared with the wild type. During light-dark transients the PEPCK mutant plants show both increased overall stomatal conductance and less responsiveness of the stomata to darkness than the wild type, indicating that stomata get 'jammed' in the open position. These results show that malate metabolism is important during dark-induced stomatal closure and that PEPCK is involved in this process.

Regulation of Dark-Induced Stomatal Closure in Arabidopsis Dynamin-Like Protein 1E (adl1e) Mutant Leaves

Arabidopsis thaliana dynamin-like protein 1E (ADL1E) is known to regulate mitochondrial elongation. The adl1e mutant has no morphological phenotype, and the growth and photosynthetic activity of the mutant are similar to those of the wild type. Leaf O2 uptake, which is supported by mitochondrial activity in the dark, is increased 1.7-fold by mutation in adl1e gene. The ATP content in the dark of guard and mesophyll cell protoplasts (GCPs and MCPs, respectively) was 2.5- to 4-fold higher in GCPs of the mutant and the wild type, and increased upon the addition of glucose in both genotypes. Oligomycin, an inhibitor of mitochondrial ATPase, suppressed ATP synthesis in both GCPs and MCPS isolated from adl1e plants, indicating that mutant had higher mitochondrial activity. The stomatal apertures of mutant and wild-type plants were then analyzed in vitro. In the light, the stomata of both genotypes showed similar patterns of opening. However, in the dark response, the stomata of the adl1e mutant closed faster than did those of the wild type. Oligomycin severely inhibited dark-induced stomatal closure in both cell types. The results suggest that stomatal closure in the dark is governed by cytosolic ATP concentration, which is stimulated by mitochondrial activity.

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.

Fusicoccin inhibits dark-induced stomatal closure by reducing nitric oxide in the guard cells of broad bean

By using pharmacological approaches and laser scanning confocal microscopy based on 4,5-diaminofluorescein diacetate (DAF-2DA), the relationship between the inhibition of dark-induced stomatal closure caused by fusicoccin (FC) and the changes of nitric oxide (NO) levels in guard cells in broad bean was studied. The results show that, like 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), a NO scavenger and NG-nitro-L-Arg-methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), FC inhibited stomatal closure induced by darkness, and reduced the levels of NO in guard cells in darkness, indicating that FC inhibits dark-induced stomatal closure through lessening NO levels in guard cells. In addition, similar to c-PTIO, both FC and butyric acid not only suppressed sodium nitroprusside (SNP)-induced stomatal closure and DAF-2DA fluorescence in guard cells, but also reopened the closed stomata induced by dark and removed NO that had been generated by dark. The results show that both FC and butyric acid cause NO removal in guard cells, and also suggest that FC-caused NO removal is probably associated with cytosolic acidification in guard cells. Taken together, our results show that FC perhaps causes cytosolic acidification in guard cells, consequently induces NO removal and reduces NO levels in guard cells, and finally inhibits stomatal closure induced by dark.

Role and interrelationship of PTPases and H2O2 in light/dark-regulated stomatal movement in Vicia faba

Role and interrelationship of protein tyrosine phosphatases (PTPases) and H2O2 in light/dark-regulated stomatal movement in Vicia faba were investigated by epidermal strip bioassay, laser-scanning confocal microscopy and assays of PTPase activity. Our results indicate that phenylarsine oxide (PAO), a specific inhibitor of PTPases, ascorbic acid (ASA), an important reducing substrate for H2O2 removal, and catalase (CAT), one of the H2O2 scavenging enzymes, did not cause any change of stomatal aperture in light, but remarkably prevented dark-induced stomatal closure. Exogenous H2O2 had no obvious effect on stomatal aperture in the dark, but significantly induced stomatal closure in light. Both PTPase activity in epidermal strips and endogenous H2O2 level in guard cells in the dark were higher than those in light. The results showed that both PTPases and H2O2 mediate light/dark-regulated stomatal movement, that dark-induced stomatal closure requires the activation of PTPases and the enhancement of H2O2 levels in guard cells, and stomatal opening caused by light is associated with the inactivation of PTPases and the reduction of H2O2 levels in guard cells. Additionally, like ASA and CAT, PAO abolished dark-, exogenous H2O2-induced stomatal closure and dichlorofluorescein fluorescence in guard cells, indicating that activation of PTPases can enhance H2O2 levels probably via suppressing the decrease of H2O2 levels in guard cells. On the other hand, similar to PAO, ASA and CAT evidently prevented dark-, exogenous H2O2-induced stomatal closure and obviously inactivated PTPases in the dark. However, exogenous H2O2 significantly activated PTPases in light. The results show that H2O2 can induce activation of PTPases. Taken together, the present results provide evidence that both H2O2 and PTPases are involved in light/dark-regulated stomatal movement, and the interaction between H2O2 and PTPases plays a pivotal role in light/dark signal transduction process in guard cells.

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.

Pharmacological evidence indicates that MAPKK/CDPK modulate NO levels in darkness-induced stomatal closure of broad bean

By using pharmacological approaches and laser scanning confocal microscopy (LSCM) based on 4, 5-diaminofluorescein diacetate (DAF-2 DA), the roles of MAPKK/CDPK and their effects on nitric oxide (NO) levels of guard cells during darkness-induced stomatal closure in broad bean were investigated. The results indicated that both 2′-amino-3′-methoxyflavone (PD98059) (an inhibitor of mitogen-activated protein kinase kinase, MAPKK) and trifluoperazine (TFP) (a specific inhibitor of calcium-dependent protein kinase, CDPK) reduced the levels of NO in guard cells and significantly reversed darkness-induced stomatal closure, implying that MAPKK/CDPK mediate darkness-induced stomatal closure by enhancing NO levels in guard cells. In addition, as with NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), but not with nitric oxide synthase inhibitor NG-nitro-L-Arg-methyl ester (L-NAME), PD98059 and TFP not only reduced 4,5-diaminofluorescein diacetate (DAF-2 DA) fluorescence in guard cells by sodium nitroprusside (SNP) in light, but also abolished NO that had been generated during a dark period, and reversed stomatal closure by SNP and by darkness, suggesting MAPKK and CDPK are probably related to restraining the NO scavenging to elevate NO levels in guard cells, during darkness-induced stomatal closure. The results also showed that both PD98059 and TFP reduced stomatal closure by SNP, implying that the possibility of MAPKK and CDPK acting as the target downstream of NO should not be ruled out. There may be a causal and interdependent relationship between MAPKK/CDPK and NO in darkness-induced stomatal closure, and in the process this cross-talk may lead to the formation of a self-amplification loop about them.

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.

Guard Cells: Transcription Factors Regulate Stomatal Movements

Recent work shows that transcription factors are necessary for stomatal movements in plants. Different members of the plant-specific R2R3-MYB transcription factor family are required for mediating stomatal opening in response to light and stomatal closure in response to darkness.

Diacylglycerols induce both ion pumping in patch-clamped guard-cell protoplasts and opening of intact stomata.

Stomatal guard cells in leaves regulate the apertures of microscopic pores through which photosynthetic gas exchange and water vapor loss occur. Environmental signals, including light, high humidity, and low CO2 concentrations, open stomata by increasing the volume of guard cells. Activation of a plasma membrane H+ pump initiates K+ and Cl- influx, accompanied by malate synthesis, resulting in osmotic water flow into the guard cells, a bowing apart of the guard-cell pair, and consequent stomatal opening. Physiological and electrophysiological techniques were employed to investigate the possibility that a second-messenger lipid, 1,2-diacylglycerol, is involved in the transduction of opening stimuli. The synthetic diacylglycerols 1,2-dihexanoylglycerol and 1,2-dioctanoylglycerol enhanced light-induced stomatal opening in Commelina communis and induced stomatal opening under darkness, whereas an isomer with no known second-messenger role, 1,3-dioctanoylglycerol, did not affect stomatal responses. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, the enzyme typically activated by 1,2-diacylglycerol in animal cells, inhibited light-stimulated stomatal opening and enhanced dark-induced stomatal closure. N-[(2-Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), which inhibits cyclic nucleotide-dependent protein kinases preferentially over lipid-dependent protein kinases such as protein kinase C, had little effect on stomatal apertures. Whole-cell patch clamping of guard-cell protoplasts of Vicia faba revealed that 1,2-dihexanoylglycerol and 1-oleoyl-2-acetylglycerol activated an ATP-dependent, voltage-independent current, suggesting activation of an electrogenic ion pump such as the H+ pump. Diacylglycerol or functionally similar lipids may act through protein phosphorylation to provide the intracellular signals that mediate H+-ATPase activation and stomatal opening in response to light or other opening stimuli.

Role of Ca2+ and EGTA on Stomatal Movements in Commelina communis L.

Ca(2+) (0.1-1.0 millimolar) accelerated dark-induced stomatal closure and reduced stomatal apertures in the light in epidermal peels of Commelina communis L. In contrast, ethyleneglycol-bis-(beta-aminoethyl ether) N,N'tetraacetic acid (EGTA) (2 millimolar), a Ca(2+) chelator, prevented closure in the dark and accelerated opening in the light. EGTA did not promote significant opening in the dark. It is therefore concluded that EGTA does not increase ion uptake into guard cells, but rather prevents ion efflux. Addition of EGTA to incubating solutions with 10 millimolar KCl resulted in steady state apertures of 15.6 micrometers, whereas in the absence of EGTA similar apertures required 55 millimolar KCl and 150 millimolar KCl was needed in the presence of 1 millimolar CaCl(2). The results demonstrate the importance of Ca(2+) in the regulation of stomatal closure and point to a role of Ca(2+) in the regulation of K(+) efflux from stomatal guard cells.