Functional Study Of A Homologous NAC Transcription Factor In Canola And Arabidopsis Regulating Reactive Oxygen Species Accumulation And Cell Death

Oilseed rape（Brassica napus L.）is the third largest oilseed crop in the world,accounting for 13%of global vegetable oil production.Both abiotic and biotic stresses reduce the yield and quality of rape seed.Therefore,studying the molecular mechanism of rapeseed in response to adverse environment is beneficial to breed stress tolerant oilseed rape.As a gene family specific to plants,NAC transcription factor genes have important biological functions in the regulation of plant growth and development,abiotic and biotic stress tolerance.In this paper,the functions and molecular mechanisms of homologous NACa in Brassica napus and Arabidopsis thaliana were studied.First,we performed subcellular localization of AtNACa and found that it was located in the nucleus.Histochemical staining was performed and the results demonstrated AtNACa is expressed in roots and veins of the seedlings,calyces,siliques and pollens of mature plants.To further study the function of AtNACa,we obtained the inducible expression lines of AtNACa and found that overexpression of AtNACa can affect the growth and development of seedlings,with significantly shorter roots and smaller leaves.Then,we conducted phenotypic assay of inducible overexpression lines of AtNACa under oxidative and osmotic stresses as well as natural aging test.The results showed that the AtNACa overexpression lines demonstrated,to some extent,tolerance to oxidative and osmotic stresses.After treated with methyl viologen（MV）and mannitol,the root elongation percentages of overexpression plants were higher than that of the GFP-expressing plants.There was no difference in leaf size between the over-expression and the control lines.This may be due to the fact that AtNACa induces the production of ROS in vivo,and ROS at a suitable concentration range may help plants get acclimated to stress conditions.This rendered AtNACa overexpression lines more tolerant to stress than the control plants.In the senescence assay,we observed that the AtNACa overexpression lines promoted leaf senescence,and 3,3’-diaminobenzidine（DAB）staining showed an increase in hydrogen peroxide（H₂O₂）accumulation,decreased chlorophyll content and increased ion permeability.These data support that overexpression of AtNACa can induce excessive accumulation of reactive oxygen species（ROS）and promote leaf senescence.Finally,we performed a dual luciferase assay of ROS,senescence,and cell death-related marker genes.We found that AtNACa activated transcription of two genes that encode bifunctional nuclease 1 and C2H2-type zinc finger family protein 12,respectively.The transcript levels of BnaNACa in oilseed rape leaves of different ages were detected by qRT-PCR.The results showed that the expression level of BnaNACa was lower at the seedling stage,maturation stage,and early senescence stage.The expression level of BnaNACa increased significantly at the late stage of leaf senescence.This suggests that BnaNACa might play an important role at the late stage of leaf senescence.Transient expression of BnaNACa in rapeseed protoplasts followed by 2’,7’-dichlorodihy-drofluorescein diacatate（H₂DCFDA）and fluorescein diacetate（FDA）staining showed that BnaNACa can promote ROS accumulation and cell death.Through dual-LUC analysis,it was demonstrated that BnaNACa can activate downstream ROS production,cell death,senescence and defense-related marker gene expression.Therefore,BnaNACa may act as a positive regulator of ROS metabolism and cell death,promoting ROS accumulation and cell death.Therefore,we explored the function of a homologous NACa transcription factor in canola and Arabidopsis,and analyzed the molecular mechanisms of ROS and cell death,providing a theoretical basis for genetic breeding of rapeseed.