Hydrogen sulfide （H2S） is a newly discovered gaseous signaling molecule and involved in ethylene and ABA-induced stomatal closure. As an important factor, extracellular ATP （eATP） was believed to participate in regulation of stomatal closing. However, the mechanism by which eATP mediates HES-regulated stomatal closure remains unclear. Here, we employed Arabidopsis wild-type and mutant lines of ATP-binding cassette transporters （Atmrp4, Atmrp5 and their double mutant Atmrp4/5） to study the function of eATP in H_2S-regulated stomatal movement. Our results indicated that H_2S affected stomatal closing through stimulating guard cell outward K^＋ current. Moreover, we found that HES induced eATP generation by regulating the activity of an ABC transporter. The inhibitor of ABC transporters, glibenclamide （Gli）, could impair H_2S-regulated stomatal closure and reduce H_2S-dependent eATP accumulation in Atmrp4 and Atmrp5 mutants. In addition, the promotion effect of H_2S on outward K^＋ currents was diminished in Atmrp4/5 double mutant. Our data suggested that hydrogen peroxide （H_2O_2） is required for H_2S-induced stomatal closure, and the production of H_2O_2 is regulated by eATP via NADPH oxidase. Based on this work, we conclude that H_2S-induced stomatal closure requires ABC transporter-dependent eATP pro- duction and subsequent NADPH oxidase-dependent H_2O_2 accumulation.
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 Viciafaba 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 II. 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.
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 (pyridoxal-phosphate-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.
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 （H2S） 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, H2S 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 H2S generated from D-/L-CDes may play an important role in ethylene-induced stomatal closure. Meanwhile, H2S synthetic inhibitors significantly inhibited ethylene-induced stomatal closure in Arabidopsis. Ethylene treatment caused an increase of H2S 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 H2S is involved in the regulation of ethylene-induced stomatal closure in Arabidopsis thaliana.
