Dark-induced Stomatal Closure Research Articles

COP1 Mediates Dark-Induced Stomatal Closure by Suppressing FT, TSF and SOC1 Expression to Promote NO Accumulation in Arabidopsis Guard Cells

RING-finger-type ubiquitin E3 ligase Constitutively Photomorphogenic 1 (COP1) and floral integrators such as FLOWERING LOCUS T (FT), TWIN SISTER OF FT (TSF) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) have been identified as regulators of stomatal movement. However, little is known about their roles and relationship in dark-induced stomatal closure. Here, we demonstrated that COP1 is required for dark-induced stomatal closure using cop1 mutant. The cop1 mutant closed stomata in response to exogenous nitric oxide (NO) but not hydrogen peroxide (H2O2), and H2O2 but not NO accumulated in cop1 in darkness, further indicating that COP1 acts downstream of H2O2 and upstream of NO in dark-induced stomatal closure. Expression of FT, TSF and SOC1 in wild-type (WT) plants decreased significantly with dark duration time, but this process was blocked in cop1. Furthermore, ft, tsf, and soc1 mutants accumulated NO and closed stomata faster than WT plants in response to darkness. Altogether, our results indicate that COP1 transduces H2O2 signaling, promotes NO accumulation in guard cells by suppressing FT, TSF and SOC1 expression, and consequently leads to stomatal closure in darkness. These findings add new insights into the mechanisms of dark-induced stomatal closure.

Transcriptome profiling of Arabidopsis slac1-3 mutant reveals compensatory alterations in gene expression underlying defective stomatal closure.

Plants adjust their stomatal aperture for regulating CO2 uptake and transpiration. S-type anion channel SLAC1 (slow anion channel-associated 1) is required for stomatal closure in response to various stimuli such as abscisic acid, CO2, and light/dark transitions etc. Arabidopsis slac1 mutants exhibited defects in stimulus-induced stomatal closure, reduced sensitivity to darkness, and faster water loss from detached leaves. The global transcriptomic response of a plant with defective stimuli-induced stomatal closure (particularly because of defects in SLAC1) remains to be explored. In the current research we attempted to address the same biological question by comparing the global transcriptomic changes in Arabidopsis slac1-3 mutant and wild-type (WT) under dark, and dehydration stress, using RNA-sequencing. Abscisic acid (ABA)- and dark-induced stomatal closure was defective in Arabidopsis slac1-3 mutants, consequently the mutants had cooler leaf temperature than WT. Next, we determined the transcriptomic response of the slac1-3 mutant and WT under dark and dehydration stress. Under dehydration stress, the molecular response of slac1-3 mutant was clearly distinct from WT; the number of differentially expressed genes (DEGs) was significantly higher in mutant than WT. Dehydration induced DEGs in mutant were related to hormone signaling pathways, and biotic and abiotic stress response. Although, overall number of DEGs in both genotypes was not different under dark, however, the expression pattern was very much distinct; whereas majority of DEGs in WT were found to be downregulated, in slac1-3 majority were upregulated under dark. Further, a set 262 DEGs was identified with opposite expression pattern between WT and mutant under light–darkness transition. Amongst these, DEGs belonging to stress hormone pathways, and biotic and abiotic stress response were over-represented. To sum up, we have reported gene expression reprogramming underlying slac1-3 mutation and resultantly defective stomatal closure in Arabidopsis. Moreover, the induction of biotic and abiotic response in mutant under dehydration and darkness could be suggestive of the role of stomata as a switch in triggering these responses. To summarize, the data presented here provides useful insights into the gene expression reprogramming underlying slac1-3 mutation and resultant defects in stomatal closure.

Hydrogen sulfide induced by hydrogen peroxide mediates darkness-induced stomatal closure in Arabidopsis thaliana

Hydrogen sulfide induced by hydrogen peroxide mediates darkness-induced stomatal closure in Arabidopsis thaliana

ABA signalling and metabolism are not essential for dark-induced stomatal closure but affect response speed

Stomata are microscopic pores that open and close, acting to balance CO2 uptake with water loss. Stomata close in response to various signals including the drought hormone abscisic acid (ABA), microbe-associated-molecular-patterns, high CO2 levels, and darkness. The signalling pathways underlying ABA-induced stomatal closure are well known, however, the mechanism for dark-induced stomatal closure is less clear. ABA signalling has been suggested to play a role in dark-induced stomatal closure, but it is unclear how this occurs. Here we investigate the role of ABA in promoting dark-induced stomatal closure. Tracking stomatal movements on the surface of leaf discs we find, although steady state stomatal apertures are affected by mutations in ABA signalling and metabolism genes, all mutants investigated close in response to darkness. However, we observed a delayed response to darkness for certain ABA signalling and metabolism mutants. Investigating this further in the quadruple ABA receptor mutant (pyr1pyl1pyl2pyl4), compared with wild-type, we found reduced stomatal conductance kinetics. Although our results suggest a non-essential role for ABA in dark-induced stomatal closure, we show that ABA modulates the speed of the dark-induced closure response. These results highlight the role of ABA signalling and metabolic pathways as potential targets for enhancing stomatal movement kinetics.

Downregulating VAC14 in Guard Cells Causes Drought Hypersensitivity by Inhibiting Stomatal Closure.

Stomata are a key land plant innovation that permit the regulation of gaseous exchanges between the plant interior and the surrounding environment. By opening or closing, stomata regulate transpiration of water though the plant; and these actions are coordinated with acquisition of CO2 for photosynthesis. Stomatal movement is controlled by various environmental and physiological factors and associates with multiple intracellular activities, among which the dynamic remodeling of vacuoles plays a crucial role. Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] is critical for dynamic remodeling of vacuoles. Its production requires a PI(3,5)P2-metabolizing complex consisting of FAB1/PIKfyve kinases, SAC phosphatases, and the scaffolding protein VAC14. Although genetic or pharmacological downregulation of PI(3,5)P2 causes hyposensitivity to ABA-induced stomatal closure, whether the effect of PI(3,5)P2 on stomatal movement is cell-autonomous and the physiological consequences of its reduction were unclear. We report that downregulating Arabidopsis VAC14 specifically in guard cells by artificial microRNAs (amiR-VAC14) results in enlarged guard cells and hyposensitivity to ABA- and dark-induced stomatal closure. Vacuolar fission during stomatal closure is compromised by downregulating VAC14 in guard cells. Exogenous application of PI(3,5)P2 rescued the amiR-VAC14 phenotype whereas PI(3,5)P2 inhibitor YM201636 caused wild-type plants to have inhibited stomatal closure. We further show that downregulating VAC14 specifically in guard cells impairs drought tolerance, suggestive of a key role of guard cell-produced PI(3,5)P2 in plant fitness.

Blue Light Improves Stomatal Function and Dark-Induced Closure of Rose Leaves (Rosa x hybrida) Developed at High Air Humidity.

Plants developed under constant high (>85%) relative air humidity (RH) have larger stomata that are unable to close completely in response to closing stimuli. Roses (Rosa x hybrida) developed in high RH have previously been shown to have high water loss during leaf dehydration and reduced dark-induced closure resulting in a shorter postharvest life. In this study, the effect of B-light on stomatal function under high RH conditions was investigated. The ability of rose leaves developed under continuous high (90%) or moderate (60%) RH to close their stomata in response to darkness and leaf dehydration assay was studied. Moreover, the level and regulation of ABA in light and darkness in relation to B-light was measured. Our results show that increased B-light proportion improved stomatal function and dark-induced stomatal closure under high RH conditions and that was associated with increased [ABA] in general and a dynamic ABA peak during darkness. Furthermore, increased B-light during the day was associated with the presence of high β-glucosidase activity during night. This indicates that B-light is important as a signal to activate the β-glucosidase enzyme and release ABA during night. Altogether, the improved stomatal function and reduced transpiration in combination with increased [ABA] indicate that preharvest B-light plays an important role in governing stomatal functionality and ABA homeostasis under high RH and can be a useful method to improve postharvest water balance of roses.

The cytosolic protein GRP1 facilitates abscisic acid- and darkness-induced stomatal closure in Salvia miltiorrhiza

By screening an expressed sequence tag (EST) library of Salvia miltiorrhiza, we detected an acidic protein, SmGRP1, with no significant similarities to the other sequences in public databases. SmGRP1 encodes a peptide of 151 amino acids, 33.77 % of which are glutamic acid residues, and the peptide was positive according to “stains-all” staining. Expression analysis revealed that SmGRP1 was expressed in all examined tissues of S. miltiorrhiza but was most highly expressed in the leaves and stems. Without a signal peptide, SmGRP1 localized to the cytoplasm in protoplasts in subcellular localization experiments. SmGRP1 expression was prominently enhanced by ABA and darkness treatments; the protein could also be induced by high temperature, NaCl, and dehydration treatments, while low temperature suppressed its expression. Furthermore, although there were no obvious phenotypic differences in SmGRP1 overexpression and SmGRP1 knockdown mutants compared with control plants under normal culture conditions, the stomata of the knockdown lines remained open when treated with ABA, darkness, NO, and H2O2. In addition, the water loss rate of the knockdown mutants was faster than that of the control lines and overexpression mutants when exposed to air. These observations indicate that SmGRP1 is a novel acidic protein with potential calcium-binding capability and is involved in stomatal movement and stress resistance.

Expression profiling of genes encoding ABA route components in response to dehydration or various light conditions in poplar buds and leaves

In this report, the members of PP2C, SnRK2a and Rboh oxidase families from Arabidopsis and poplar were studied in silico, and the expression profiles of the some of them were specified in Populus tremula buds and adult leaves. In poplars, the counterparts of ABI1- and ABI2-like protein phosphatases are lacking, but poplar genomes encode three HAB-like proteins denoted in this work as HAB1, HAB3a and HAB3b, and the counterparts of the two latter ones are absent in Arabidopsis. Nonetheless, they may be present in other species. In poplars, SnRK2 subclass III includes two SnRK2.6-like protein kinases denoted by us as SnRK2.6a and SnRK2.6b, and only one SnRK2.2 corresponding to SnRK2.2 and SnRK2.3 ones from Arabidopsis. In contrast to Arabidopsis, the poplar Rboh family involves two RbohD- and RbohF-like proteins denoted here as RbohD1 and RbohD2, and RbohF1 and RbohF2, respectively.The expressions of genes encoding the above components of the ABA route were studied in Populus tremula dehydrated buds and adult leaves not subjected to stress but exposed to natural daylight or to darkness, and to inhibition of ethylene biosynthesis or signaling route by cobalt or silver ions, respectively. In leaves, the light conditions seemed to be the most pronounced factor, from among the studied stimuli, controlling the expression Ptre-HAB3a, Ptre-HAB1, Ptre-SnRK2.6a and Ptre-RbohF2 genes, their expression was upregulated in darkness. This observation implies that these genes may be important for dark-induced stomatal closure regulation. Ethylene negatively affected the expression of three studied Rboh genes and Ptre-HAB1one but only at daylight, whereas its positive effect on the of Ptre-HAB3a was shown in the dark exposed leaves.In buds, three studied Rboh genes took part in the early response to dehydration, however their participation involved the visibly highest level of the Ptre-RbohD1 transcripts, followed by Ptre-RbohF2 and the lowest one of Ptre-RbohF1. Nonetheless, the further stress-induced superoxide anion generation seemed to depend on the enhanced expression of the Ptre-RbohD1 and Ptre-RbohF2 genes only, still with a significantly higher level of the Ptre-RbohD1 one. Ptre-RbohD2 transcripts were found neither in leaves nor in buds. The expression of the other genes discussed in the present work was either slightly upregulated at moderate stress or did not significantly change in response to dehydration. The protein kinase activity of overexpressed Ptre-SnRK2.6a and Ptre-SnRK2.6b was confirmed in in vitro protein kinase assay and compared to that of SnRK2.6/OST1 one from Arabidopsis.

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.

Regulation of stomatal movement by cortical microtubule organization in response to darkness and ABA signaling in Arabidopsis

Microtubule dynamics are essential for plant cell development and in producing responses to external stimuli. However, little is known about the regulation of microtubule dynamics or crosstalk between microtubule and stomatal movement. Here we identified microtubule reorganization as a crucial factor determining guard cell responses to dark and abscisic acid (ABA) signaling. As stomata opened, guard cells exhibited radially arranged cortical microtubules, which depolymerized into the cytosol when exposed to darkness and ABA. Suppression of microtubule disassembly by paclitaxel, a microtubule-stabilizing drug, significantly enhanced stomatal aperture under light, and partially blocked ABA- or darkness-induced stomatal closure. However, treatment with only the anti-microtubule drug, oryzalin, did not affect stomatal movement with or without external stimuli. Phosphatidic acid (PA) bound to a clade A type 2C protein phosphatase (PP2C), PP2CA, and deletion of PP2CA partially inhibited PA-induced microtubule depolymerization and stomatal closure. Moreover, microtubule reorganization was altered in the ABA-insensitive mutant pldα1, but not in the ABA-hypersensitive mutant pp2ca. We propose that a faithfully balanced reorganization of microtubules fulfills fundamental functions to enable the fast change of stomata in plant adaptive responses to developmental and environmental cues.

PtoMYB170 positively regulates lignin deposition during wood formation in poplar and confers drought tolerance in transgenic Arabidopsis

Wood formation is a complex developmental process under multi-level transcriptional control executed by a large set of transcription factors. However, only limited members have been characterized to be key regulators of lignin biosynthesis in poplar. Here we report the conserved and unique functions of PtoMYB170, a transcription factor identified from Populus tomentosa (Chinese white poplar), in lignin deposition and drought tolerance in comparison with its duplicate paralog PtoMYB216. PtoMYB170 is preferentially expressed in young leaves and xylem tissues. Overexpression of PtoMYB170 in transgenic poplar plants resulted in stronger lignification and more thickened secondary wall in xylem compared with wild-type plants, whereas the CRISPR/Cas9-generated mutation of PtoMYB170 weakened lignin deposition, thereby leading to a more flexible and collapsed xylem phenotype. Transient expression experiments demonstrated that PtoMYB170 specifically activated the expression of lignin biosynthetic genes, consistent with the function of PtoMYB216. However, GUS staining assays revealed that PtoMYB170 was specifically expressed in guard cells of transgenic Arabidopsis while PtoMYB216 was not. Heterologous expression of PtoMYB170 in Arabidopsis enhanced stomatal closure in the dark and resulted in drought tolerance of the transgenic plants through reduced water loss, indicating a diversified role from PtoMYB216. These results revealed the PtoMYB170-dependent positive transcriptional regulation on lignin deposition in poplar and its coordinated function in enhancing drought tolerance by promoting dark-induced stomatal closure.

Role and interrelationship of MEK1-MPK6 cascade, hydrogen peroxide and nitric oxide in darkness-induced stomatal closure

Pharmacological data have suggested the involvement of mitogen-activated protein kinase (MPK) cascades in dark-induced stomatal closure, but which specific MPK cascade participates in the darkness guard cell signaling and its relationship with hydrogen peroxide (H2O2) and nitric oxide (NO) remain unclear. In this paper, we observed that darkness induced activation of MPK6 in leaves of wild-type Arabidopsis (Arabidopsis thaliana) and mutants for nitrate reductase 1 (NIA1), but this effect was inhibited in mutants for MPK Kinase 1 (MEK1) and ATRBOHD/F. Mutants for MEK1, MPK6 and NIA1 showed defect of dark-induced NO production in guard cells and stomatal closure, but were normal in the dark-induced H2O2 generation, while stomata of mutant AtrbohD/F showed defect of dark-induced H2O2 and NO production and subsequent closure. Moreover, exogenous NO rescued the defect of dark-induced stomatal closure in mutants of AtrbohD/F, mek1 and mpk6, while exogenous H2O2 could not rescue the defect of dark-induced stomatal closure in mutants of mek1, mpk6 and nia1. These genetic and biochemical evidences not only show that MEK1-MPK6 cascade, AtRBOHD/F-dependent H2O2 and NIA1-dependent NO are all involved in dark-induced stomatal closure in Arabidopsis, also indicate that MEK1-MPK6 cascade functions via working downstream of H2O2 and upstream of NO.

The role of phytosphingosine-1-phosphate (Phyto-S1P) and its relationships with cytosolic pH and hydrogen peroxide (H2O2) during stomatal closure by darkness in broad bean

We examined the role of phytosphingosine-1-phosphate (Phyto-S1P) and its relationships with cytosolic pH and hydrogen peroxide (H2O2) during stomatal closure by darkness using broad bean as experimental material. Darkness caused cytoplasmic alkalization, induced H2O2 generation, and led to stomatal closure. DL-threo-dihydrosphingosine (DL-threo-DHS) and N,N-dimethylsphingosine (DMS) inhibited these effects of darkness. Exogenous Phyto-S1P promoted cytoplasmic alkalization, H2O2 synthesis, and caused stomatal closure. Butyric acid largely suppressed cytoplasmic alkalization and prevented stomatal closure by Phyto-S1P. The H2O2 modulators ascorbic acid (ASA), catalase (CAT), and diphenyleneiodonium (DPI) obviously restricted Phyto-S1P-promoted H2O2 synthesis and stomatal closure. These results suggest that Phyto-S1P was involved in darkness-induced stomatal closure by causing cytoplasmic alkalization and H2O2 synthesis. Furthermore, darkness-induced H2O2 synthesis was inhibited by butyric acid, suggesting that cytoplasmic alkalization caused by Phyto-S1P was probably necessary for H2O2 synthesis. The rise in cytosolic pH promoted by Phyto-S1P became noticeable at 6min and reached a maximum at 18min, while H2O2 levels sharply increased at 8min and peaked at 22min, and butyric acid prevented Phyto-S1P-promoted H2O2 synthesis, which confirmed the conclusion that Phyto-S1P-induced cytosolic alkalization acts upstream of the production of H2O2 during stomatal closure by darkness. We suggest that Phyto-S1P is involved in stomatal closure by darkness through cytoplasmic alkalization and H2O2 synthesis, and Phyto-S1P-promoted cytoplasmic alkalization acts upstream of H2O2 synthesis in the process.

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

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.

Differential Role of Ethylene and Hydrogen Peroxide in Dark-induced Stomatal Closure

Regulation of stomatal aperture is crucial in terrestrial plants for controlling water loss and gaseous exchange with environment. While much is known of signaling for stomatal opening induced by blue light and the role of hormones, little is known about the regulation of stomatal closing in darkness. The present study was aimed to verify their role in stomatal regulation in darkness. Epidermal peelings from the leaves of Commelina benghalensis were incubated in a defined medium in darkness for 1 h followed by a 1 h incubation in different test solutions [H2O2, propyl gallate, ethrel (ethylene), AgNO3, sodium orthovanadate, tetraethyl ammonium chloride, CaCl2, LaCl3, separately and in combination] before stomatal apertures were measured under the microscope. In the dark stomata remained closed under treatments with ethylene and propyl gallate but opened widely in the presence of H2O2 and AgNO3. The opening effect was largely unaffected by supplementing the treatment with Na-vanadate (PM H+ ATPase inhibitor) and tetraethyl ammonium chloride (K(+)-channel inhibitor) except that opening was significantly inhibited by the latter in presence of H2O2. On the other hand, H2O2 could not override the closing effect of ethylene at any concentrations while a marginal opening of stomata was found when Ag NO3 treatment was given together with propyl gallate. CaCl2 treatment opened stomata in the darkness while LaCl3 maintained stomata closed. A combination of LaCl3 and propyl gallate strongly promoted stomatal opening. A probable action of ethylene in closing stomata of Commelina benghalensis in dark has been proposed.

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.

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....

Sphingosine-1-phosphate (S1P) mediates darkness-induced stomatal closure through raising cytosol pH and hydrogen peroxide (H2O2) levels in guard cells in Vicia faba

The role and signaling of sphingosine-1-phosphate (S1P) during darkness-induced stomatal closure were examined in Vicia faba. Darkness substantially raised S1P and hydrogen peroxide (H(2)O(2)) levels and closed stomata. These darkness effects were significantly suppressed by DL-threo-dihydrosphingosine (DL-threo-DHS) and N,N-dimethylsphingosine (DMS), two inhibitors of long-chain base kinases. Exogenous S1P led to stomatal closure and H(2)O(2) production, and the effects of S1P were largely prevented by the H(2)O(2) modulators ascorbic acid, catalase, and diphenyleneiodonium. These results indicated that S1P mediated darkness-induced stomatal closure by triggering H(2)O(2) production. In addition, DL-threo-DHS and DMS significantly suppressed both darkness-induced cytosolic alkalization in guard cells and stomatal closure. Exogenous S1P caused cytosolic alkalization and stomatal closure, which could be largely abolished by butyric acid. These results demonstrated that S1P synthesis was necessary for cytosolic alkalization during stomatal closure caused by darkness. Furthermore, together with the data described above, inhibition of darkness-induced H(2)O(2) production by butyric acid revealed that S1P synthesis-induced cytosolic alkalization was a prerequisite for H(2)O(2) production during stomatal closure caused by darkness, a conclusion supported by the facts that the pH increase caused by exogenous S1P had a shorter lag and peaked faster than H(2)O(2) levels and that butyric acid prevented exogenous S1P-induced H(2)O(2) production. Altogether, our data suggested that darkness induced S1P synthesis, causing cytosolic alkalization and subsequent H(2)O(2) production, finally leading to stomatal closure.

Effect of PLC on Stomatal Movement in <i>Vicia faba L.</i>

The effect of PLC in dark-regulated stomatal movement in Vicia faba L. was studied by means of pharmacological approach and stomatal analysis. The result shows that dark-induced stomatal closure can be blocked by U73122, which is a kind of special inhibitor for PLC, and so does the NOs inhibitor, L-NAME and the scavenger of NO, c-PTIO. So this result indicated that the bioactivity of PLC was high in darkness, and some relationship may lie between PLC and NO in stomatal movement.