Ethylene-induced Stomatal Closure Research Articles

GCR1 Positively Regulates UV-B- and Ethylene-Induced Stomatal Closure via Activating GPA1-Dependent ROS and NO Production.

Heterotrimeric G proteins function as key players in guard cell signaling to many stimuli, including ultraviolet B (UV-B) and ethylene, but whether guard cell G protein signaling is activated by the only one potential G protein-coupled receptor, GCR1, is still unclear. Here, we found that gcr1 null mutants showed defects in UV-B- and ethylene-induced stomatal closure and production of reactive oxygen species (ROS) and nitric oxide (NO) in guard cells, but these defects could be rescued by the application of a Gα activator or overexpression of a constitutively active form of Gα subunit GPA1 (cGPA1). Moreover, the exogenous application of hydrogen peroxide (H2O2) or NO triggered stomatal closure in gcr1 mutants and cGPA1 transgenic plants in the absence or presence of UV-B or ethylene, but exogenous ethylene could not rescue the defect of gcr1 mutants in UV-B-induced stomatal closure, and gcr1 mutants did not affect UV-B-induced ethylene production in Arabidopsis leaves. These results indicate that GCR1 positively controls UV-B- and ethylene-induced stomatal closure by activating GPA1-dependent ROS and NO production in guard cells and that ethylene acts upstream of GCR1 to transduce UV-B guard cell signaling, which establishes the existence of a classic paradigm of G protein signaling in guard cell signaling to UV-B and ethylene.

Ethylene-induced stomatal closure is mediated via MKK1/3-MPK3/6 cascade to EIN2 and EIN3.

Mitogen-activated protein kinases (MPKs) play essential roles in guard cell signaling, but whether MPK cascades participate in guard cell ethylene signaling and interact with hydrogen peroxide (H2 O2 ), nitric oxide (NO), and ethylene-signaling components remain unclear. Here, we report that ethylene activated MPK3 and MPK6 in the leaves of wild-type Arabidopsis thaliana as well as ethylene insensitive2 (ein2), ein3, nitrate reductase1 (nia1), and nia2 mutants, but this effect was impaired in ethylene response1 (etr1), nicotinamide adenine dinucleotide phosphate oxidase AtrbohF, mpk kinase1 (mkk1), and mkk3 mutants. By contrast, the constitutive triple response1 (ctr1) mutant had constitutively active MPK3 and MPK6. Yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays indicated that MPK3 and MPK6 physically interacted with MKK1, MKK3, and the C-terminal region of EIN2 (EIN2 CEND). mkk1, mkk3, mpk3, and mpk6 mutants had typical levels of ethylene-induced H2 O2 generation but impaired ethylene-induced EIN2 CEND cleavage and nuclear translocation, EIN3 protein accumulation, NO production in guard cells, and stomatal closure. These results show that the MKK1/3-MPK3/6 cascade mediates ethylene-induced stomatal closure by functioning downstream of ETR1, CTR1, and H2 O2 to interact with EIN2, thereby promoting EIN3 accumulation and EIN3-dependent NO production in guard cells.

Ethylene-Induced Hydrogen Sulfide Negatively Regulates Ethylene Biosynthesis by Persulfidation of ACO in Tomato Under Osmotic Stress.

A number of recent studies identified hydrogen sulfide (H2S) as an important signal in plant development and adaptation to environmental stress. H2S has been proven to participate in ethylene-induced stomatal closure, but how the signaling pathways of H2S and ethylene interact is still unclear. Here, we reveal how H2S controls the feedback-regulation of ethylene biosynthesis in tomato (Solanum lycopersicum) under osmotic stress. We found that ethylene induced the production of H2S in guard cells. The supply of hypotaurine (HT; a H2S scavenger) or DL-pro-pargylglycine (PAG; a synthetic inhibitor of H2S) removed the effect of ethylene or osmotic stress on stomatal closure. This suggests that ethylene-induced H2S is a downstream component of osmotic stress signaling, which is required for ethylene-induced stomatal closure under osmotic stress. We further found that H2S inhibited ethylene synthesis through inhibiting the activity of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidases (ACOs) by persulfidation. A modified biotin-switch method (MBST) showed that H2S can induce persulfidation of LeACO1 and LeACO2 in a dose-dependent manner, and that persulfidation inhibits the activity of LeACO1 and LeACO2. We also found that LeACO1 is persulfidated at cysteine 60. These data suggested that ethylene-induced H2S negatively regulates ethylene biosynthesis by persulfidation of LeACOs. In addition, H2S was also found to inhibit the expression of LeACO genes. The results provide insight on the general mode of action of H2S and contribute to a better understanding of a plant’s response to osmotic stress.

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English

Heterotrimeric G protein mediates ethylene-induced stomatal closure via hydrogen peroxide synthesis in Arabidopsis.

Heterotrimeric G proteins function as key players in hydrogen peroxide (H2O2) production in plant cells, but whether G proteins mediate ethylene-induced H2O2 production and stomatal closure are not clear. Here, evidences are provided to show the Gα subunit GPA1 as a missing link between ethylene and H2O2 in guard cell ethylene signalling. In wild-type leaves, ethylene-triggered H2O2 synthesis and stomatal closure were dependent on activation of Gα. GPA1 mutants showed the defect of ethylene-induced H2O2 production and stomatal closure, whereas wGα and cGα overexpression lines showed faster stomatal closure and H2O2 production in response to ethylene. Ethylene-triggered H2O2 generation and stomatal closure were impaired in RAN1, ETR1, ERS1 and EIN4 mutants but not impaired in ETR2 and ERS2 mutants. Gα activator and H2O2 rescued the defect of RAN1 and EIN4 mutants or etr1-3 in ethylene-induced H2O2 production and stomatal closure, but only rescued the defect of ERS1 mutants or etr1-1 and etr1-9 in ethylene-induced H2O2 production. Stomata of CTR1 mutants showed constitutive H2O2 production and stomatal closure, but which could be abolished by Gα inhibitor. Stomata of EIN2, EIN3 and ARR2 mutants did not close in responses to ethylene, Gα activator or H2O2, but do generate H2O2 following challenge of ethylene or Gα activator. The data indicate that Gα mediates ethylene-induced stomatal closure via H2O2 production, and acts downstream of RAN1, ETR1, ERS1, EIN4 and CTR1 and upstream of EIN2, EIN3 and ARR2. The data also show that ETR1 and ERS1 mediate both ethylene and H2O2 signalling in guard cells.

Extracellular Adenosine Triphosphate Functions Downstream of Hydrogen Sulfide in Ethylene-induced Stomatal Closure in Arabidopsis thaliana

Extracellular Adenosine Triphosphate Functions Downstream of Hydrogen Sulfide in Ethylene-induced Stomatal Closure in <i>Arabidopsis thaliana</i>

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.

Involvement of Copper Amine Oxidase (CuAO)-Dependent Hydrogen Peroxide Synthesis in Ethylene-Induced Stomatal Closure inVicia faba

Involvement of Copper Amine Oxidase (CuAO)-Dependent Hydrogen Peroxide Synthesis in Ethylene-Induced Stomatal Closure in<i>Vicia faba</i>

Hydrogen Sulfide Regulates Ethylene‐induced Stomatal Closure in Arabidopsis thaliana

Hydrogen sulfide (H2 S) is a newly-discovered signaling molecule in plants and has caused increasing attention in recent years, but its function in stomatal movement is unclear. In plants, H2 S is synthesized via cysteine degradation catalyzed by D-/L-cysteine desulfhydrase (D-/L-CDes). AtD-/L-CDes::GUS transgenic Arabidopsis thaliana (L.) Heynh. plants were generated and used to investigate gene expression patterns, and results showed that AtD-/L-CDes can be expressed in guard cells. We also determined the subcellular localization of AtD-/L-CDes using transgenic plants of AtD-/L-CDes::GFP, and the results showed that AtD-CDes and AtL-CDes are located in the chloroplast and in the cytoplasm, respectively. The transcript levels of AtD-CDes and AtL-CDes were affected by the chemicals that cause stomatal closure. Among these factors, ACC, a precursor of ethylene, has the most significant effect, which indicates that the H2 S generated from D-/L-CDes may play an important role in ethylene-induced stomatal closure. Meanwhile, H2 S synthetic inhibitors significantly inhibited ethylene-induced stomatal closure in Arabidopsis. Ethylene treatment caused an increase of H2 S production and of AtD-/L-CDes activity in Arabidopsis leaves. AtD-/L-CDes over-expressing plants exhibited enhanced induction of stomatal closure compared to the wild-type after ethylene treatment; however, the effect was not observed in the Atd-cdes and Atl-cdes mutants. In conclusion, our results suggest that the D-/L-CDes-generated H2 S is involved in the regulation of ethylene-induced stomatal closure in Arabidopsis thaliana.

Regulatory Function of Polyamine Oxidase-Generated Hydrogen Peroxide in Ethylene-Induced Stomatal Closure in Arabidopsis thaliana

Hydrogen peroxide (H2O2) is an important signaling molecule in ethylene-induced stomatal closure in Arabidopsis thaliana. Early studies on the sources of H2O2 mainly focused on NADPH oxidases and cell-wall peroxidases. Here, we report the involvement of polyamine oxidases (PAOs) in ethylene-induced H2O2 production in guard cells. In Arabidopsis epidermal peels, application of PAO inhibitors caused the failure of ethylene to induce H2O2 production and stomatal closure. Results of quantitative RT-PCR analysis and pharmacological experiments showed that AtPAO2 and AtPAO4 transcripts and activities of PAOs were both induced by ethylene. In transgenic Arabidopsis plants over-expressing AtPAO2 and AtPAO4, stomatal movement was more sensitive to ethylene treatment and H2O2 production was also significantly induced. The increased H2O2 production in the transgenic lines compared to the wild-type plants suggests that AtPAO2 and AtPAO4 probably are involved in ethylene-induced H2O2 production. Several factors which induce stomatal closure such as dehydration and high salinity all enhanced the expression of AtPAO2 and AtPAO4 to different degrees. Moreover, GFP-AtPAOs fusion protein localized in the nucleus, cytoplasm, and cell wall of the guard cells. Therefore, our results strongly indicated that PAO is a source of H2O2 generation in Arabidopsis guard cells and plays crucial roles in stomatal movement.

Hydrogen Sulfide May Function Downstream of Nitric Oxide in Ethylene-Induced Stomatal Closure in Vicia faba L.

Pharmacological, laser scanning confocal microscopic (LSCM), and spectrophotographic approaches were used to study the roles of hydrogen sulfide (H2S) and nitric oxide (NO) in signaling transduction of stomatal movement in response to ethylene in Vicia faba L. Ethylene treatment resulted in the dose-dependent stomatal closure under light, and this effect was blocked by the inhibitors of H2S biosynthesis in V. faba L. Additionally, ethylene induces H2S generation and increases L-/D-cysteine desulfhydrase (pyridoxalphosphate-dependent enzyme) activity in leaves of V. faba L. Inhibitors of H2S biosynthesis have no effect on the ethylene-induced stomatal closure, NO accumulation, and nitrate reductase (NR) activity in guard cells or leaves of V. faba L. Moreover, the ethylene-induced increase of H2S levels and L-/D-cysteine desulfhydrase activity declined when NO generation was inhibited. Therefore, we conclude that H2S and NO probably are involved in the signal transduction pathway of ethylene-induced stomatal closure. H2S may represent a novel component downstream of NO in the ethylene-induced stomatal movement in V. faba L.

Identification of two-component system elements downstream of AHK5 in the stomatal closure response of Arabidopsis thaliana

To optimize water use efficiency, plants regulate stomatal closure through a complex signaling process. Hydrogen peroxide (H2O2) is produced in response to several environmental stimuli, and has been identified as a key second messenger involved in the regulation of stomatal aperture. The Arabidopsis histidine kinase 5 (AHK5) has been shown to regulate stomatal closure in response to H2O2 and other stimuli that depend on H2O2. AHK5 is a member of the two-component system (TCS) in Arabidopsis. The plant TCS comprises three different protein types: the hybrid histidine kinases (HKs), the phosphotransfer proteins (HPs) and the response regulators (RRs). Here we determined TCS elements involved in H2O2- and ethylene-dependent stomatal closure downstream of AHK5. By yeast and in planta interaction assays and functional studies, AHP1, 2 and 5 as well as the response regulators ARR4 and ARR7 were identified acting downstream of AHK5 in the ethylene and H2O2 response pathways of guard cells. Furthermore, we demonstrate that aspartate phosphorylation of ARR4 is only required for the H2O2- but not for the ethylene-induced stomatal closure response. Our data suggest the presence of a complex TCS signaling network comprising of at least AHK5, several AHPs and response regulators, which modulate stomatal closure in response to H2O2 and ethylene.

Hydrogen sulfide induced by nitric oxide mediates ethylene-induced stomatal closure of Arabidopsis thaliana

Pharmacological, laser scanning confocal microscopic (LSCM), real-time PCR and spectrophotographic approaches are used to study the roles of hydrogen sulfide (H2S) and nitric oxide (NO) in signaling transduction of stomatal movement response to ethylene in Arabidopsis thaliana. In the present study, inhibitors of H2S synthesis were found to block ethylene-induced stomatal closure of Arabidopsis. Treatment with ethylene induced H2S generation and increased L-/D-cysteine desulfhydrase (pyridoxalphosphate-dependent enzyme) activity in leaves. Quantitative PCR analysis showed AtL-CDes and AtD-CDes transcripts were induced by ethylene. It is suggested that ethylene-induced H2S levels and L-/D-cysteine desulfhydrase activity decreased when NO was compromised. The data clearly show that ethylene was able to induce H2S generation and stomatal closure in Atnoa1 plants, but failed in the Atnia1,nia2 mutant. Inhibitors of H2S synthesis had no effect on ethylene-induced NO accumulation and nitrate reductase (NR) activity in guard cells or leaves of Arabidopsis, whereas ethylene was able to induce NO synthesis. Therefore, we conclude that H2S and NO are involved in the signal transduction pathway of ethylene-induced stomatal closure. In Arabidopsis, H2S may represent a novel downstream indicator of NO during ethylene-induced stomatal movement.

Ethylene-induced nitric oxide production and stomatal closure in Arabidopsis thaliana depending on changes in cytosolic pH

We investigated changes in cytosolic pH and nitric oxide (NO) during ethylene-induced stomatal closure in Arabidopsis thaliana using pharmacological, laser scanning confocal microscopy (LSCM), and spectrophotography techniques. Treatment with ethephon (a direct source of ethylene when applied to plants) and 1-aminocycloaminopropane-1-carboxylic acid (ACC, an ethylene precursor) resulted in a rapid accumulation of NO and cytosolic alkalinization in guard cells. Acetic acid (a weak acid) and sodium orthovanadate (NaVO3; a plasmalemma H+-ATPase inhibitor) reduced stomatal closure induced by ethylene and blocked ethylene-induced activity of nitrate reductase. However, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a NO scavenger, had no effect. These results suggest that NO production is downstream of the rise in cytosolic pH in A. thaliana.

The interplay between redox and hormone signalling in Arabidopsis guard cells

There is now much evidence that the reactive oxygen species (ROS) hydrogen peroxide (H2O2) plays an important role in a number of physiological responses, including stomatal closure. H2O2 is generated in stomatal guard cells in response to drought stress, increased concentrations of abscisic acid (ABA), ozone and also in response to elicitor/pathogen challenge. Generation of H2O2 occurs via the AtrbohD/F subunits of the NADPH oxidase complex. In recent work we have shown that the plant hormone ethylene, known to influence numerous processes such as fruit ripening, senescence, defence responses and seedling growth, also mediates stomatal closure in intact Arabidopsis leaves. Using a combination of genetics and physiological tools, we have demonstrated that ethylene-induced stomatal closure is dependent on H2O2 synthesis via AtrbohF. Guard cells of the ethylene receptor mutants etr1–1 and etr1–3 are insensitive to ethylene, but not to ABA. Moreover stomata of the ethylene signalling ein2-1 and arr2 mutants do not close in response to ethylene, but do generate H2O2 following ethylene treatment. In addition, we have also observed that whilst ethylene alone can cause closure, ethylene in the presence of ABA does not cause stomatal closure in leaves. These data demonstrate that plant hormones mediate their effects in guard cells via complex interplay with redox signalling pathways.