The Mechanism Exploration Of Vacuolar Na~+/H~+ Antiporter(NHX1) In Regulating Metal Ion Transportation And Resistance Enhancement In Tobacco

Nowadays,a large area of the soil is found contaminated with excess metal due to anthropogenic activities,which threatens the physiological metabolism and yield of plants.It is therefore emergent to improve the metal tolerance of plant for enhancing plant growth and phytoremediation efficiency.The expression of vacuolar Na~+/H~+antiporter 1(NHX1)could be induced by multi metal stresses,which was also proven to elevate the excess Na~+tolerance of plants.However,researches on the association between NHX1 and plant tolerance to other metal ionsremains limited except for Na~+stress.In the present work,the relationship between metal tolerance and NHX1 gene was elucidated by using Ss NHX1 gene from Suaeda salsa,Se NHX1 gene from Salicornia europaea,and Sse NHX1 gene obtained by DNA family shuffling taking Se NHX1 and Ss NHX1 as sequence templates.The potential mechanisms involved in this process were also discussed in this work.The relationship between NHX1 and Cd stress was firstly analyzed by adding NHX1 inhibitor and overexpressing NHX1 gene in tobacco.Results showed that tobaccos treated with NHX1 inhibitor+Cd exhibited higher MDA level,lower biomass and chlorophyll content comparing with only Cd-treated plants.Furthermore,the overexpressing of NHX1(including Ss NHX1 gene,Se NHX1 gene and Sse NHX1gene)gene enhanced the Cd resistance of tobacco seedlings comparing to non-transgenic plants.The above results preliminarily indicated that NHX1 gene might be involved in resisting Cd stress of tobacco plants.To further explore the connection between NHX1 and Cd tolerance,the physiological and biochemical changes of NHX1-trasngenic(Ss NHX1 gene,Se NHX1gene and Sse NHX1 gene)tobacco leaves were measured after the exposure to Cd stress.Results showed that,under Cd stress,NHX1-transgenic tobaccos exhibited more biomass production,greater chlorophyll content,higher photosynthetic capacity,and lower accumulation of ROS and MDA as compared with non-transgenic tobaccos.Higher Na~+and K~+content,and lower Cd accumulation were also found in transgenic tobacco leaves comparing to non-transgenic plants when exposed to Cd stress.Moreover,the Cd tolerance of Sse NHX1-overexpressing tobacco was significantly higher than Ss NHX1-or Se NHX1-overexpressing tobacco plants.Further analysis was conducted on Sse NHX1-overexpressing tobaccos to investigate mechanisms underlying the enhanced Cd tolerance of NHX1-transgenic tobacco plants.Results showed that the community of endophytic bacteria in NHX1-overexpressing tobacco roots was found extinguished with non-transgenic tobacco roots at genus level under Cd stress.Sphingomonas,Steroidobacter and Actinoplanes might make a significant contribution to the enhanced Cd tolerance of NHX1-trnsgenic tobaccos.Comparing to WT plants,the level of oxidative damage and Cd content werefound decreased in NHX1-transgenic tobacco root under Cd exposure.Also,enzyme activities of rhizosphere soil around transgenic tobacco roots were higher than that of WT tobaccos.These results suggested that the changes on endophytic bacterial community of NHX1-transgenic tobacco roots might enhance Cd tolerance of tobacco by improving enzyme activities of rhizosphere soil,strengthening antioxidant defense system of toabcco roots,and maintaining ion content of tobacco plants.The role of NHX1 in resisting different valence metal stress(Li(I),Pb(II),Cr(III)and Cr(VI))was then studied in Sse NHX1-overexpressing tobaccos.Results showed that,when exposed to Li(I),Pb(II),Cr(III)or Cr(VI)stress,the translocation factor from roots to leaves of NHX1-transgenic tobacco was found lower than non-transgenic tobacco plants.The content of Li,Pb or Cr in vacuoles was found increased in NHX1-transgenic tobacco roots under Li(I),Pb(II),Cr(III)or Cr(VI)stress.The tolerance of NHX1-transgenic tobacco to different valence metal stress decreased gradually with the increase of metal ion valence from I to VI.In conclusion,this study indicated that the metal resistance in NHX1-transgenic tobaccos was associated with the increase of rhizosphere soil enzyme activities,changes of endophytic bacteria community and decrease of oxidative damage level,resulting from the regulation of the accumulation and transportation of metal ions in NHX1-overexpressing tobaccos under different metal stresses.This study provided a theoretical basis for the tolerance mechanism of NHX1 gene in regulating different metal ion stresses and had practical significance for improving phytoremediation efficiency.