The Role Of H₂S In Darkness-induced Stomatal Closure And Its Relationship With H₂O₂

Many researchs have shown that hydrogen sulfide（H₂S）and hydrogen peroxide（H₂O₂）are important components involved in the process of signal transduction in plants.However,The effect of H₂S on the darkness-induced stomatal closure and its relationship with H₂O₂ are not clear.In the Present work,We used Vicia faba L.,Arabidopsis wild type（col-0）and its H₂S and H₂O₂ mutants（atl-cdes,atl-cdes,atrbohD,atrbohF,atrbohD/F）as the materials and investigated by using pharmacological,spectrophotographic and lasers canning confocal microscopic approaches to study the problems.The results are as follows:1.We examined the effects of NaHS,which is the donor of H₂S,on stomatal aperture in V.faba.The results showed that treatment with≥0.05 mmol·L^-1 NaHS significantly caused stomatal closure within a 1.5h.Elution test results showed that the effects of NaHS(0.05 and0.1 mmol·L^-1)on stomatal movement were reversible.These results illustrated that H₂S is effective for inducing stomatal closure and in a dose-dependent manner.2.Darkness-induced stomatal closure is partially blocked by H₂S scavenger or inhibitors in V.faba and A.thaliana wide type,and the content of H₂S and the activity of L-/D-CDes were increased significantly in darkness.Besides,there was no obvious effects on stomatal aperture of the H₂S synthetic mutant atl-cdes and atd-cdes in darkness,these results implied that H₂S is involved in darkness-induced stomatal closure.3.After treatment with 3h under different concentrations of H₂O₂,Treatment with≥100μmol·L^-1 H₂O₂ significantly induced stomatal closure of V.faba in light.It was indicated that exogenous H₂O₂ could induce the stomatal closure in V.faba and in a dose-dependent manner.4.Darkness could induce H₂O₂ accumulation in guard cells of V.faba and A.thaliana.However,the increase of H₂O₂ levels and the darkness-induced stomatal closure was inhibited by H₂O₂ scavenger and inhibitors.In addition,Darkness could induce stomatal closure in wild type and H₂O₂ synthetic mutant atrbohD,however,there was no significant change in stomatal aperture of the H₂O₂ synthetic mutant atrbohF and atrbohD/F in darkness.These suggested that H₂O₂ is involved in darkness-induced stomatal closure.5.H₂O₂ scavenger and synthesis inhibitors had no effects on NaHS-induced stomatal closure in V.faba.However,H₂S content and L-/D-CDes activity were significantly decreased after application with the H₂O₂-scavenging enzyme CAT or inhibitors ASA,DPI and SHAM in darkness.And in contrast,there is almost no effects on H₂O₂ levels in darkness when in presence of the H₂S scavenger HT or H₂S synthesis inhibitors NH₂OH,AOA and C₃H₃KO₃+NH₃.Besides,there was no significant effects on the H₂S content and L-/D-CDes activity in atrbohF and atrbohD/F,but the leves of H₂O₂ increased in guard cells of atl-cdes and atd-cdes.From these data it can be deduced that H₂S is involved in darkness-induced stomatal closure and acts downstream of H₂O₂ in V.faba.