The Role Of Ethylene In Abscisic Acid And Dark Induced Stomatal Closure And Its Relationship To Ethylene Signaling Pathways And H 2 O 2

Ethylene is a plant hormone that plays important roles in regulation of plant growth,developmental processes,responses of external biological and abiotic stress stimuli,as well as many aspects of physiological activities of plants.Interestingly,many studies showed that ethylene has dual effects on the regulation of stomatal movement:it not only can induce stomatal closure solely,but also can inhibit ABA-and dark-induced stomatal closure.At present,there are a lot of researches about the signal transduction mechanism of ethylene-induced stomatal closure.However,the phenomenon that ethylene inhibits ABA-and dark-induced stomatal closure,are still under debate,and the understanding of the mechanism are still limited.Further comparison of previous inconsistent reports.We find that there are differences in the use of materials,the concentration of exogenous ethylene and the composition of the buffer.In this report,using Arabidopsis?Arabidopsis thaliana?wild-type Col-0 and Ws as well as the mutants for ethylene perception and signaling etrl-1,etrl-9,ersl,ersl-3,ein4,ein2-1,ein2-5,ein3-1,arr2-3,arr2-4,and ethylene over-production mutant eto3,we comparatively studied the effects of endogenous and exogenous ethylene in Ca²⁺ and Ca²⁺-free buffers on ABA-and dark-induced stomatal closure and their relationships between ethylene signaling-pathway and signal molecule H₂O₂.The main results and conclusions were as following:1.10 ?M ABA could induce stomatal closure of the ethylene over-production mutant eto3 in both Ca²⁺ and Ca²⁺-free buffer,but the stomatal closing speed of eto3 was significantly slower than wild type.And ethylene bio-synthesis inhibitor AVG could markedly accelerate the rate of ABA-induced wild-type stomatal closure.Also ABA-induced stomatal closure of mutants for ethylene perception and signaling was faster than wild type,and the differences was most significant at 15 min after ABA treatment.In addition,the H₂O₂ level of guard cells of ein3-1 mutant was significantly higher than that wild type at 15 min after ABA treatment.The results suggest that endogenous ethylene can reduce the level of H₂O₂ in guard cells by ethylene signaling pathway,and thus inhibit ABA-induced stomatal closure.2.In the Ca²⁺-buffer 10 ?M ACC could obviously inhibit stomatal closure induced by ABA in wild-type Arabidopsis,but it could not inhibit ABA-induced stomatal closure of mutants for ethylene perception and signaling.And exogenous ACC treatment could clearly reduce the H₂O₂ level of wild type Arabidopsis thaliana guard cells under ABA treatment,but it had no effect on the level of H₂O₂ in ein3-1 mutant guard cells under ABA treatment.The results showed that exogenous ethylene inhibited ABA-induced stomatal closure by reducing the level of H₂O₂ in the guard cells through the ethylene signaling pathway Meanwhile,in the Ca²⁺-free buffer,the exogenous ACC treatment had no significant effects on ABA-induced stomatal closure,indicating that the effects of exogenous ethylene on ABA-induced stomatal closure is dependent on the presence of Ca²⁺ in the buffer.3.In the Ca²⁺-buffer,10 ?M ACC markedly promoted the re-opening of wild-type stomata that had been induced to close by ABA,but 10 ?M ACC did not promote the re-opening of stomata of mutants of ethylene signaling that had been induced to close by ABA.And ACC treatment could reduce the level of H₂O₂ generated by ABA in wild-type guard cells,but it had no effect on the level of H₂O₂ generated by ABA in ein3-1 guard cells,suggesting that ethylene removed H₂O₂ in guard cells induced by ABA through the ethylene signaling pathway,and thus allowed the closed stomata to be reopened.In the Ca²⁺-free buffer,exogenous ACC could not induce the re-opening of stomata that had been induced to close by ABA,indicating that the exogenous ethylene promotes the re-opening of stomata that had been induced to close by ABA,depended on the presence of Ca²⁺ in the buffer.4.In the Ca²⁺ and Ca²⁺-free buffer,dark could induce stomatal closure of the ethylene over-production mutant eto3,but the stomatal closure speed of eto3 was distinctly slower than the wild type.And ethylene bio-synthesis inhibitor AVG could speed up the rate of dark-induced wild-type stomatal closure;Dark-induced stomatal closure of mutants for ethylene perception and signaling was faster than wild type,the level of endogenous H₂O₂ in ein3-1 mutant accumulate faster than that in wild type.The above results suggest that endogenous ethylene reduces the level of H₂O₂ in the guard cell through the ethylene signaling pathway and thus inhibits dark-induced stomatal closure.5.In the Ca²⁺-free buffer,500 ?M ACC could markly inhibit stomatal closure induced by darkness treatment in wild-type Arabidopsis,and 500?M ACC could also suppress dark-induced stomatal closure of mutants for ethylene perception and signaling.Meanwhile,exogenous ACC treatment could evidently inhibit the accumulation of H₂O₂ in Arabidopsis thaliana wild-type guard cells,and reduce the accumulation of H₂O₂ induced by dark in ein3-1 guard cells,These results showed that exogenous ethylene inhibits dark-induced stomatal closure by reducing the level of H₂O₂ in guard cells,and this effect of exogenous ethylene does not depend on the ethylene signaling pathway.In the Ca²⁺buffer,exogenous ethylene had no significant effect on dark-induced stomatal closure.6.In a Ca²⁺-free buffer,500 ?M ACC re-opened the closed stomata of wild type caused by dark,as well as the closed stomata of mutants for ethylene perception and signaling.Meanwhile ACC reduced the level of H₂O₂ that had been generated by dark in Arabidopsis thaliana wild-type guard cells,and ACC also reduced the level of H₂O₂ generated by dark in guard cell of ein3-1.These results suggested that ACC induced the removal of H₂O₂ that had been generated by dark in guard cells,and therefore re-opened the closed stomata induced by dark,but this effect of ethylene is independent of the ethylene signal pathway.However,in the Ca²⁺ buffer,the exogenous ethylene did not re-open the closed stomata induced by dark.In summary,our results showed that endogenous ethylene inhibited ABA and dark-induced stomatal closure by ethylene signal transduction pathway,and this inhibition is related to the removal of H₂O₂ in the guard cells through ethylene signal transduction pathway.And exogenous ethylene can also inhibit the ABA and dark-induced stomatal closure of Arabidopsis thaliana by reducing the level of H₂O₂ in guard cells,but the mechanisms of reducing the levels of endogenous H₂O₂ and inhibit ABA-and dark-induced stomatal closure are different:?1?low concentration of exogenous ethylene only inhibited the ABA-induced stomatal closure,and the effect was dependent on the ethylene signal transduction pathway and Ca²⁺ in the buffer;?2?high concentrations of exogenous ethylene inhibited the effect of dark-induced stomatal closure,and its effect did not depend on the ethylene signal transduction pathway,and more importantly,ethylene had this effect only in the Ca²⁺-free buffer.