Molecular Mechanism Of Wheat Transcription Factor TaNTL5 Involved In Saline-Alkali Stress

At present,soil salinization is more and more serious to affect the global crop yield.The alkalized soil usually has higher pH value,and shows much severe impact on crop yield than saline soil.So far,much attention has been paid on the regulatory pathways of plants' responses to salinity stress,but few studies were focused on alkalinity stress tolerance mechanisms.Therefore,elucidating the molecular mechanism of plant tolerance to alkali stress and breeding new alkali-tolerant crop varieties has become an important approach to alleviate global food security problems.A series of wheat introgression lines were generated by asymmetric somatic hybridization between common wheat JN177 and tall wheatgrass in our lab,and the introgression line-SR4 was selected from them which display strong tolerance to alkalinity.Through transcriptome analysis,we identified an alkalinity responsive NAC transcription factor gene TaNTL5 from SR4 and further analyzed its role and mechanisms for alkali stress.It has been previously reported that NTLs can respond to a variety of abiotic stresses.We transformed TaNTL5 and two truncated sequences(TaNTL5-?TM and TaNTL5-?C)into Arabidopsis thaliana and observed the phenotypes of Col-0 and transgenic Arabidopsis under control and various abiotic stress conditions.We found that the phenotypes of TaNTL5-?TM transgenic lines showed no significant difference with that of wild type under control conditions and abiotic stresses.This suggested that TaNTL5 does not play a role in plant responses to abiotic stress if only the transmembrane domain was truncated.However,both TaNTL5 and TaNTL5-AC transgenic lines were tolerant to alkali stress while seneitive to salinity stress.Moreover,the TaNTL5 and TaNTL5-?C transgenic lines showed a completely opposite phenotype under both ABA and ROS stress treatment.These results suggested that TaNTL5 may be involved in plant responses to a variety of abiotic stresses,with complicated mechanisms.TaNTL5 is a type of membrane-bound transcription factor(MTFs),and releasing into the nucleus by shearing process is essential for its nuclear localization and transcriptional activation activity.The results of subcellular localization and yeast one hybrid analysis showed that the full-length TaNTL5 protein was mainly localized on the ER membrane and could not exhibit transcriptional activation activity.While TaNTL5 was released from the ER membrane to the nucleus after cutting off the transmembrane domain(TM).However,TaNTL5-?TM showed no transcriptional activation activity due to the regulation of a NAC repression domain S100 which located in the middle of TaNTL5.After cutting off S100,the truncated TaNTL5 has demonstrated transcriptional activation activity,however,some of the truncated TaNTL5 proteins localized to the protoplast membrane in addition to the nucleus.Further analysis showed that this changes may be related to S53(the first half in S100),suggesting that S100 is a key motif for regulating nuclear localization and transcriptional activity of TaNTL5.Analysis on the ABA and ROS signaling pathway genes indicated that TaNTL5 and TaNTL5-AC can up-regulate the expression of the ABA signaling pathway gene ABI5 and ROS synthesis gene RbohC.Chromatin immunoprecipitation(ChIP),electrophoretic mobility shift assay(EMSA),and GUS reporter gene experiments further confirmed this regulatory relationship.As overexpression of AtABI5 can significantly enhance the sensitivity to salt stress in Arabidopsis thaliana,TaNTL5 may enhance the salt sensitivity of plants by affecting the expression of AB15.TaNTL5 can significantly enhance resistance to alkali stress in Arabidopsis thaliana,and TaNTL5 OE lines had higher content of superoxide anion than Col-0 under both control and alkali stress treatment,which is consistent with TaNTL5 up-regulation of ROS synthesis gene RbohC,suggesting that RbohC may play an important role in TaNTL5-mediated alkali stress response in plant.