Exploration Of Function And Molecular Mechanisms Of Two NAC Transcription Factors Regulating ROS Accumulation And Leaf Senescence In Oilseed Rape

Oilseed rape（Brassica napus.L）is one of the most important oil crops in the world.Its yield and quality are affected by a variety of biotic and abiotic factors.Reactive oxygen species（ROS）play an important role in pathogen defence and aging.During senescence,leaf cells carry out programmed cell death,accompanied by nutrients relocating from sencent leaves to young leaves or storage organs,especially in seeds,which are critical for crop yield.NAC（NAM,ATAF1/2,CUC2）transcription factor family is specific to plants.Regulatory mechenism of NAC TFs in ROS accumulation and leaf senescence are poorly understood in oilseed rape.In this study,two NAC transcription factor genes BnaNAC87 and BnaNAC60were cloned from Brassica napus L.,and both of them containing a conserved NAC domain.There is a transmembrane domain in the C-terminus of BnaNAC60.Subcellular location assay was performed to examine their expression location in plant cells.The expression pattern of these two NAC TFs during leaf development and stress treatments were analyzed through q RT-PCR.Phenotype and physiological indexes were detected after the overexpression of these two genes in tobacco leaves,protoplasts and arabidopsis.The putative target genes were screened by q RT-PCR and Dual-LUC assay,and confirmed by EMSA and Ch IP-q PCR in vitro and in vivo.BnaNAC87 is a transcriptional activator and localized in the nuclei.The expression of BnaNAC87 was significantly increased in late senescent leaves,and its expression was induced by salt,dehydration,ABA,salicylic acid,jasmonic acid and S.sclerotiorum.BnaNAC60 is a membrane-tethered NAC transcription factor associated with endoplasmic reticulum（ER）membrane and ER stress can induce its relocation from ER membrane to nuclei.Its truncated form without transmembrane domain BnaNAC60ΔTM is located in nuclei.BnaNAC60 is highly expressed in the early senescent leaves,late senescent leaves and tip of the early senescent leaves.Expression of BnaNACA60 can be induced by salt,dehydration,ABA and cold.The cis-elements for BnaNAC87 is the NAC recognition site（NACRS）containing the core CATGTG motif,which is a canonical element for SNAC subfamily.BnaNAC60 binds to a novel cis-element but not NACRS,namely NTLBS containing the core TTNCTT motif,which is a specific motif for membrane-tethered NAC transcription factor.Transient expression of BnaNAC87 in tobacco leaves induced HR-like cell death and ROS accumulation,which lead to chlorophyll degradation,anthocyanin and MDA accumulation,relative conductivity increase and severe DNA fragment.Overexpression of BnaNAC87 in oilseed rape protoplasts induced ROS accumulation and cell death as well.Transient overexpression of the full length BnaNAC60 and its truncated form BnaNAC60ΔTM in tobacco leaves induced cell death,and the syptom in BnaNAC60ΔTM expressing tissues is more serious than that in BnaNAC60 expressing tissues.Physiological indexes were significantly changed in these tissues,including ROS accumulation,chlorophyll degradation,increase of anthocyanin and MDA content.Transient expression of BnaNAC60and BnaNAC60ΔTM in protoplasts of oilseed rape induced cell death and ROS accumulation,and the percent of dead protoplasts in BnaNAC60ΔTM overexpressed cells is much higher than that of the full length BnaNAC60.Transgenic arabidopsis plants overexpressing BnaNAC60ΔTM showed accelerated leaf senescence compared with the control plants under normal growth condition,accompanied by H₂O₂ accumulation,chlorophyll degradation and relative conductivity increase.Moreover,transcription of ROS-,defense-and senescence-related genes were up-regulated in these transgenic overexpression lines.Transient overexpression of BnaNAC87 in tobacco leaves induced the expression of ROS-,cell death and defense-related genes,including Nb Rboh B,Nb VPE1a,Nb ZEN1,Nb WRKY6,Nb ZAT12,Nb PR2,Nb PR5 and Nb HIN1.What’s more,BnaNAC87 can directly activate and bind to promoters of Nb ZEN1,Nb ZAT12,Nb HIN1 and Nb PR5,regulating the ROS accumulation and cell death.Transient overexpression of BnaNAC60ΔTM in canola seedlings induced the expression of marker genes related to ROS production,cell death and leaf senescence.The Dual-LUC assay showed that BnaNAC60 can activate promoters of Bna ZAT12,Bna Rboh D,Bna VPEs,Bna NYC1,Bna NYE1 and Bna BFN1.Moreover,EMSA and Ch IP-q PCR assay confirmed that BnaNAC60ΔTM can directly bind to NTLBS elements in promoters of Bna Rboh D,Bna BFN1,Bna NYC1 and Bna VPEs genes,which are downstream targets for BnaNAC60 in regulating ROS accumulation and leaf senescence.Transfer and remobilization of nutrients during leaf senescnece is of great significance for crop yield and quality.Our results indicate that two NAC transcription factors BnaNAC87and BnaNAC60 are positive regulators of ROS accumulation,cell death and leaf senescence by activating the expression of cognate target genes.This research hopefully provids a theoretical and practical basis for molecular assisted breeding for higher yield and better quality in oilseed rape.