Functional Study Of Two NAC Transcription Factor In Brassica Napus Regulating Reactive Oxygen Species Accumulation And Cell Death

There are various adverse factors affecting the growth,yield and quality of crops.To survive these challenges,plants have evolved numerous fine and complex mechanisms.As a major and plant-specific transcription factor family,NAC transcription factors play an important role in growth and development,response to biotic and abiotic stresses.Oilseed rape(Brassica napus)is a vital oil crop in the world and the loss of its product is severe because it often suffers from multiple stresses.At present,the function of NAC transcription factors in the oilseed rape remains largely unknown.In this study,we conducted a preliminary study on the functions of two NAC transcription factor genes in oilseed rape in inducing reactive oxygen specie(ROS)accumulation and cell death.Firstly,we isolated and cloned BnaNAC56 and BnaNACa genes from rapeseed and found that the transcriptional level of BnaNAC56 was induced by a variety of stresses and phytohormone treatment,including methyl viologen(MV),a necrotrophic fungal pathogen Sclerotinia sclerotiorum,abscisic aid(ABA),and jasmonic acid(JA),but repressed by cold treatment.The results of subcellular location and dual luciferase reporter system assay indicated that BnaNAC56 is located in nuclei and is a transcription activator.The expression of BnaNAC56 in N.benthamiana showed that it could induce ROS accumulation and hypersensitive response-like cell death.The measurements of several biological indexes related to ROS,cell death and defense response support it,such as relative conductivity,chlorophyll,H2O2 content and 3,3-diaminobenzidine(DAB)staining.Simultaneously,both TdT-medicated dUTP Nick End Labeling(TUNEL)and DNA ladder assays demonstrated nuclear DNA fragmentation occurred as a result of BnaNAC56 expression.Besides,the BnaNAC56 and GUS proteins were both detected in the leaf discs of N.benthamiana by western blot.To further determine the function of BnaNAC56,we isolated the protoplasts of Brassica napus and transfected transient plasmids harboring BnaNAC56 and GUS respectively into them through PEG4000.The results showed that the oilseed rape protoplasts expressing BanNAC56 did induce the accumulation of ROS and cell death compared with the control GUS.Furthermore,we performed real-time quantitative qRT-PCR experiment to detect the expression levels of some marker genes associated with ROS,cell death,and defense responses,and found that the transcriptional levels of several marker genes were significantly induced.The dual luciferase reporter system(Dual-LUC)assay further confirmed that ROSand cell death-related genes could be activated by BnaNAC56.The above results demonstrated that BnaNAC56 can function as a transcriptional activator of stress response and plays an important role in regulating ROS accumulation and cell death.Similarly,we transiently expressed BnaNACa in tobacco leaves and identified a similar phenomenon as compared to BnaNAC56,that is,ROS accumulation and hypersensitive response-like cell death.The result of DAB staining showed obvious accumulation of H2O2 in tobacco leaves expressing BnaNACa.Consistent with it,the determination of H2O2 content also showed it increases continually as time goes by.The measurement of MDA,anthocyanin,chlorophyll contents and relative conductivity also supported the function of BnaNACa.However,the regulatory mechanism of BnaNACa in this process is not clear and awaits further study.Overall,in this study,we identified the function of two NAC transcription factor genes which have not been reported in rapeseed,and laid the foundation for a comprehensive analysis of the regulatory mechanism of the two genes.