Functional Characterization And Preliminary Analyses Of The Underlining Regulatory Mechanisms Of Rapeseed NACa And Arabidopsis WRKYx Transcription Factor Genes

Plants are challenged with various biotic and abiotic stresses during the process of growth and development.Not only can these stresses affect the distribution of plants,but also determine the survival of plants.In the long-term evolution process,plants have evolved a variety of sophisticated mechanisms to respond to stresses and enhance adaptability.Transcription factors regulate the expression of growth-,stress-and defense-related genes to influence the survival of plants,which is an important regulatory way in plants.NAC and WRKY transcription factor gene families are two important gene families in plants.In this study,we identified a rapeseed(Brassica napus L.)NACa gene which promotes the accumulation of reactive oxygen species(ROS)and induces cell death in Nicotiana benthamiana leaves which expressing the BnaNACa and explored the underlining mechanism preliminarily.At the same time,we studied the role of an Arabidopsis thaliana WRKYx gene in response to abiotic stresses.In previous experiments,BnaNACa had been cloned successfully from rapeseed.We identified that BnaNACa protein was located in nucleus of Nicotiana benthamiana epidermal cells through Green fluorescent protein(GFP)fusion,and quantitative reverse transcription PCR(qRT-PCR)demonstrated BnaNACa responded to cold,abscisic acid(ABA)and methyl viologen(MV)treatments.In this study,we identified that BnaNACa was a transcription activator by yeast transactivational assay.Further expression assay in Nicotiana benthamiana indicated that ecotopic expression of BnaNACa induced accumulation of ROS and hypersensitive response-like cell death,which was supported by DAB(3,3'-diaminobenzidine)staining and quantitation of content of hydrogen peroxide(H2O2).The content of malondialdehyde(MDA)significantly increased in the leaf discs expressing BnaNACa,compared to those of GFP control.We also performed the DNA ladder and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)assays to examine the change of nucler DNA in leaf discs,and the results showed that expression of BnaNACa caused obvious nuclear DNA fragmentation.Moreover,the concentration of chlorophyll was significantly decreased,but the relative conductivity and the content of anthocyanins weresignificantly increased in leaf discs expressing BnaNACa.Subsequently,we identified that BnaNACa could activate the expression of ROS-and defense-related genes by qRT-PCR and dual luciferase reporter assay.Furthermore,we found that expression of BnaNACa in Arabidopsis led to acceleration of root elongation and leaf senescence when treatment with ABA,however,the exact mechanism awaits to be investigated.AtWRKYx is a WRKY transcription factor gene in Arabidopsis which was cloned successfully in previous research.The research identified that AtWRKYx was a transcriptional repressor by transcriptional activity assay in yeast.We studied the expression level of AtWRKYx in young seedlings after treated with various stresses by qRT-PCR,and the results indicated that AtWRKYx responded to cold(4oC),glucose(4%),heat(37?),salt(200mM NaCl),dehydration(12.5% PEG8000)and ABA(50 ?M ABA)treatments.We further identified homozygous T-DNA insertion mutant of AtWRKYx and subjected it to phenotypic assay at the germination level.The results showed that the germination of seeds in early stage were accelerated in the atwrkyx.Moreover,bimolecular fluorescence complementation(BiFC)and yeast two-hybrid(Y2H)experiments showed that AtWRKYx may fulfill its function through forming homo-or heterodimers.However,a detailed functional characterization of AtWRKYx remains to be done.Taken together,this study has deepened our understanding of the roles and regulatory mechanisms of NAC and WRKY transcription factor genes in plant development and stress responses and provided a solid foundation for further study.