The Role Of H₂S In Salt Stress-induced Stomatal Closure And Its Relationship With H₂O₂ And No In Arabidopsis Thaliana

In the present study,wild-type Arabidopsis thaliana L.(Col-0),nitric oxide synthase(NOS)mutant Atnoa1 and nitrate reductase(NR)mutants of Nia2-5/Nia1-2,NADPH oxidase gene mutants atrboh D,atrboh F and atrboh D/F and the L-/D-cysteine desulfhydras deletion mutants Atl-cdes and Atd-cdes were selected as plant materials.The effect of hydrogen sulfide(H₂S)in salt stress-induced stomatal movement in A.thaliana,and its relationship with hydrogen peroxide(H₂O₂)and nitric oxide(NO)were studied.The main results are as follows:1.Na Cl significantly promoted closure of the open stomata,and the optimum concentration and the optimum treatment time of Na Cl for inducing stomatal closure were100 mmol·L^-1and 1 h respectively.Elution experiments showed that the influence of Na Cl on stomatal aperture was not caused by irreversible cytotoxicity.The result suggested that Na Cl could induce stomatal closure of wild-type A.thaliana.2.H₂S scavenger,H₂S synthesis inhibitors and enzymolysis products obviously prevented Na Cl-induced stomatal closure,and Na Cl induced L-/D-CDes activity and H₂S biosynthesis were also decreased in wild-type leaf.In addition,Na Cl could induce stomatal closure in wild type,but Atl-cdes and Atd-cdes mutants showed no effect.The results showed that At L-CDes-/At D-CDes-catalyzed H₂S might be involved in salt stress-induced stomatal closure in A.thaliana.3.H₂O₂scavenger and synthesis inhibitors significantly inhibited Na Cl-induced stomatal closure in wild type.Na Cl was significantly promoted closure of the open stomata in atrboh D mutant,but atrboh F and atrboh D/F mutants showed no difference in stomatal aperture size from the wild type.Using H₂O₂specific fluorescent probe H₂DCF-DA,and observed under fluorescence microscope:Na Cl can induced strong H₂DCF fluorescence in wild-type guard cells,but it was significantly reduced after treatment with H₂O₂scavenger and synthesis inhibitors,respectively.Na Cl can increase the H₂O₂level in atrboh D mutant,but had no effect on atrboh F and atrboh D/F mutants.The results suggested that H₂O₂is involved in Na Cl-induced stomatal closure,and Atrboh F protein might be required for H₂O₂production in NADPH oxidase process in A.thaliana.4.NO scavenger and synthesis inhibitors significantly inhibited Na Cl-induced stomatal closure in the wild type.Atnoa1 and Nia2-5/Nia1-2 mutants on Na Cl was no difference in stomatal aperture size from the wild type.Using NO specific fluorescent probe DAF-2DA,and observed under fluorescence microscope:Na Cl can induced strong DAF-2fluorescence in wild-type guard cells,but it was significantly reduced after treatment with H₂O₂scavenger and synthesis inhibitors,respectively.Na Cl had not significant effect on NO level of Atnoa1 and Nia2-5/Nia1-2 mutants.The data confirmed that NO sourced from NOS and NR is involved in Na Cl-induced stomatal movement,and NO production depends on the Nia and At NOA1 pathways.5.H₂O₂scavengers and synthesis inhibitor in Na Cl stress decreased H₂S content and L-/D-CDes activity in wild type.L-/D-CDes activity and H₂S biosynthesis in leaves of wild type and atrboh D mutant after Na Cl processing was significantly higher,but had no effect on atrboh F and atrboh D/F mutants.In addition,after Na Cl and H₂S scavenger and synthesis inhibitors were respectively treated together,H₂O₂fluorescence intensity in wild-type guard cells was not weakened.The H₂O₂level of Atl-cdes and Atd-cdes mutants in leaf guard cells on Na Cl stress was no significant difference from the wild type.The results show that H₂O₂might represent a novel upstream component of H₂S signaling cascade during salt stress-induced stomatal closure in A.thaliana.6.NO scavenger and synthesis inhibitors in Na Cl stress decreased H₂S content and L-/D-CDes activity in wild type.Na Cl had no significant effect on L-/D-CDes activity and H₂S biosynthesis of Atnoa1 and Nia2-5/Nia1-2 mutants.In addition,after Na Cl and H₂S scavenger and synthesis inhibitors were respectively treated together,NO fluorescence intensity in wild-type guard cells was not weakened.The NO level of Atl-cdes and Atd-cdes mutants in leaf guard cells on Na Cl stress was no significant difference from the wild type.The results show that NO might be located upstream of H₂S during salt stress-induced stomatal closure in A.thaliana.In conclusion,These data suggested that H₂S,H₂O₂and are involved in salt stress-induced stomatal closure in A.thaliana,and H₂O_2,NO might represent a novel upstream component of H₂S signaling cascade.In the process of Na Cl-induced stomatal closure,H₂S is catalyzed by At L-CDes/At D-CDes,H₂O₂production depends on Atrboh F,and NO production depends on the Nia and At NOA1 pathways,respectively.