Molecular Mechanism Of Hydrogen Sulfide Catalyzed By L-Cysteine Desulfhydrases In Mediating Heme Oxygenase 1/Abscisic Acid-Insensitive 4 Signaling System-Induced Stomatal Movement And Drought Stress In Arabidopsis

L-cysteine desulfhydrase(DES1;EC 4.4.1.28)is an important enzyme that produces hydrogen sulfide(H2S)in plant.A large number of studies regard H2S as a gas signal molecule,which involved in a variety of physiological and biochemical signal transduction processes.But the regulation pattern of plant stomatal movement and the molecular mechanism of the regulation of drought tolerance by H2S remain unclear.Heme oxygenase(HO;EC 1.14.99.3),the rate-limiting enzyme that degrades heme to carbon monoxide(CO),and biliverdin(BV),plays a protective role against various abiotic stresses.Our previous results showed that under drought stress,between the Arabidopsis HY1 and ABI4,there occurs a plastid to nucleus retrograde HY1-ABI4 signaling pathway in the guard cells,and HY1 can negatively regulate ABI4.However,the specific signaling mechanism of this process is not clear.Whether DES1 participates in the HY1-ABI4 signaling pathway and what kind of role hydrogen sulfide plays under drought stress are not well understood.To solve the above questions,we studied the topics by pharmacological and genetic evidences,and acquired results as follows.1.H2S participates in ABA-regulated Arabidopsis stomatal movement.ABA(5 ?M)and H25 donor sodium hydrosulfide(NaHS;100 ?M could induce stomatal closure in wild-type,but ABA could not induce stomatal closure of Arabidopsis desl mutant,and the ABA-insensitive phenotype of desl mutant can be complemented by H2S donors,suggesting that H2S is involved in ABA-mediated stomatal movement;in addition,DES1 is specifically expressed in the pores of wild-type Arabidopsis.The stomata of the transgenie material pMYB60:DES1 was more sensitive than wild type in response to ABA,and endogenous H2S experiment also proved that H2S is involved in ABA-regulated stomatal movement of Arabidopsis.The relevant transgenic lines also showed a phenotype consistent with guard cells in terms of drought tolerance.2.In ABA signaling pathway,HY1 is hypersensitive to ABA and plays a negative regulatory role,located upstream of H25 in Arabidopsis guard cell.The epidermis strips of the hy1 mutant experiment revealed that when exposed to ABA treatment,the stomata closed very fast,indicating that HY1 negatively regulates ABA signaling in guard cells and is hypersensitive to ABA;while the exogenous H25 scavenger hypotaurine(HT,100 ?M)can eliminate the hypersensitivity of hy1-100 in response to ABA and the stomata of hy1-100 could not be closed,which demonstrates that HY1 is located upstream of DES1.Similarly,the hy1/des1 double mutant that removes H2S endogenously,reverses the hypersensitive phenotype of hyl.All the above demonstrated that HY1 is located upstream of H2S in Arabidopsis guard cell ABA signaling pathway.3.ABI4 located downstream of DES1 in ABA signaling.Exogenous H2S treatment to abi4 mutant revealed that H2S could not close the stomata of abi4 mutant;the transgenic line pMYB60:DES1/abi4,with specific overexpressed H2S-producing enzyme(DES1)in the guard cells,failed to respond to ABA,which is the same to abi4 mutant,indicating that the endogenous H2S could not reverse the ABA-insensitiveness of the abi4 stomata.In this case,we know that in ABA signaling pathway,ABI4 is located downstream of DES1 in Arabidopsis guard cell.In addition,related strains also have corresponding phenotypes under drought tolerance.In conclusion,in Arabidopsis guard cell ABA signaling,H2S,catalyzed by stomatal specific DES1 mediated HY1-ABI4 signaling pathway,regulates stomatal movement and drought tolerance.