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

Hydrogen sulfide?H₂S?and Hydrogen peroxide?H₂O₂?play an important role in thegrowth and development of plants.They are important components that regulate the signal transduction pathway in plants.However,the role of H₂S in BR-induced stomatal movement and the relationship between H₂S and H₂O₂ remain unclear.In this paper,Vicia faba L.,Arabidopsis thaliana L.,H₂S synthetic mutants?Atl-cdes,Atd-cdes?and H₂O₂ synthetic mutants?AtrbohD,AtrbohF,AtrbohD/F?were used as experimental materials.Pharmacological test,laser confocal microscopy and spectrophotometry were used.The role of H₂S in BR controlled stomatal closure and its relationship with H₂O₂ were examined.The experimental results are as follows:1.After the epidermal strips of broad bean leaves were treated with different concentrations of H₂S donor NaHS,it was observed that a certain concentration of NaHS could induce the stomatal closure of broad bean significantly,and showed a concentration-dependent effect.The stomatal opening of the epidermal strips could return to normal level after 1.5 h of elution treatment with 0.05 mmol·L^-11 and 0.1 mmol·L^-11 NaHS solution.The above results preliminarily proved that H₂S induced by NaHS could induce stomatal closure in V.faba.2.L-/D-cysteine desulfurase?L-/D-CDes?is the main source of H₂S in plants.The results showed that HT?scavenger of H₂S?,AOA and NH₂OH?inhibitor of L-/D-CDes?,C₃H₃KO₃+NH₃?product of L-/D-CDes reaction?can inhibit BR-induced stomatal closure of broad bean and A.thaliana wild type?Col-0?in varying degrees.In addition,BR could induce stomatal closure in A.thaliana wild-type?Col-0?,but had no significant effect on H₂S synthetic mutants Atl-cdes and Atd-cdes stomatal closure.These results indicated that H₂S was involved in BR-induced stomatal closure in V.faba and A.thaliana,the production of H₂S depends on L-/D-CDes.3.After treated with H₂O₂ of different concentrations for 3 h,it was observed that the stomatal closure of V.faba leaves could be significantly induced by H₂O₂ of more than100?mol·L^-1,and the effect was concentration dependent.The results showed that exogenous H₂O₂ could induce stomatal closure in V.faba.4.ASA?an important reducing substrate for H₂O₂ removal?,CAT?one of the H₂O₂ scavenging enzymes?,DPI?an inhibitor of H₂O₂-generating enzyme NADPH oxidase?and SHAM?an inhibitor of inhibitor of cell wall peroxidase?could effectively inhibit BR-induced stomatal closure and the increase of endogenous H₂O₂ level in V.faba,while BR-induced stomatal closure in A.thaliana wild type?Col-0?could also be inhibited by ASA,CAT,DPI and SHAM.In addition,BR could induce stomatal closure of wild type?Col-0?and single mutant AtrbohD in A.thaliana,but had no significant effect on single mutant AtrbohF and double mutant AtrbohD/F.These results suggest that BR promotes stomatal closure in V.faba and A.thaliana and is related to H₂O₂,and the generation of H₂O₂ depends on the NOX pathway.5.ASA,CAT,DPI and SHAM could decrease the content of H₂S and the activity of L-/D-CDes in BR-induced broad bean and A.thaliana wild type?Col-0?leaves.HT,AOA,NH₂OH,C₃H₃KO₃+NH₃ had no significant effect on the level of H₂O₂ in BR-induced guard cells of broad bean and A.thaliana wild type?Col-0?;BR had no significant effect on the levels of H₂O₂ in AtrbohF and AtrbohD/F leaves.The content of H₂S and the activity of L-/D-CDes were not significantly affected,and the level of H₂O₂ in guard cells of Atl-cdes and Atd-cdes was effectively increased.These results suggest that H₂S is involved in the regulation of BR-induced stomatal closure in the downstream of H₂O₂.In conclusion,H₂S is involved in the closure of BR-induced stomata of V.faba and A.thaliana wild type?Col-0?,and H₂S plays a role downstream of H₂O₂ in the process of H₂S-induced stomatal closure in V.faba and A.thaliana.In addition,the production of H₂O₂in BR-induced stomatal closure was dependent on the NOX pathway,and H₂S production was dependent on L-/D-CDes.