Dissecting The Molecular Mechanisms Of Two Transcription Factors In Arabidopsis And Oilseed Rape That Regulate Leaf Senescence And Cell Death

Leaf senescence represents the final stage of leaf growth and development,and it is critical for plants’fitness and reproduction,providing an effective way of nutrient relocation and reuse for plants.Crop traits can be improved by modulating the onset and progression of senescence which can influence the yield and quality of crops.Dissecting the regulatory mechanisms of leaf senescence can provide theoretical basis for breeding more productive and adaptable varieties.Arabidopsis thaliana,as a model plant,plays an important role in studying the molecular mechanisms of leaf senescence.At present,great advances have been achieved in understanding leaf senescence,but the underlying regulatory mechanisms remain largely unclear.Oilseed rape（Brassica napus L.）is an important oil crop in China and even in the world,but its growth and yield are often affected by various biotic and abiotic stresses.Therefore,it is crucial to identify stress-tolerant genes and reveal their functions.Transcription factors（TFs）are involved in various biological processes via regulating gene expression,and play pivotal roles in the process of crop domestication,which makes them important targets of molecular breeding of crops.This study revealed the molecular mechanism of a WRKY transcription factor in regulating the natural leaf senescence in Arabidopsis,and that of a NAC transcription factor in reactive oxygen species（ROS）accumulation and cell death in oilseed rape.Firstly,this study identified that AtWRKY42 is a senescence-related gene in Arabidopsis,and revealed its function and regulatory mechanism through molecular biology and genetic methods.The main methods and findings are as follows.1.Real time quantitative RT-PCR（qRT-PCR）analysis was conducted to examine the expression of AtWRKY42 in Arabidopsis leaves at different developmental stages.Results showed that AtWRKY42 expression was strongly induced by age and gradually increased with aging,and showed high expression in senescent leaves.2.Overexpression of AtWRKY42 dramatically accelerated the accumulation of H₂O₂and SA as well as leaf senescence process while mutation of AtWRKY42 exhibited the opposite role.Consistently,a decrease in chlorophyll content and higher relative conductivity were observed on AtWRKY42-OE lines,which demonstrated that AtWRKY42is a positive regulator in leaf senescence.3.Overexpression of AtWRKY42 promoted early flowering but had little influence on the seed yield.4.From the RNA-seq analysis of AtWRKY42 OE line and wild-type（wt）,more than2000 differentially expressed genes（DEGs）were identified,including many senescence-associated genes（SAGs）.Part of DEGs were screened with the canonical W-box（TTGACC/T）cis-element in the 1 kb promoters.Part of the DEGs were confirmed by qRT-PCR and dual-luciferase assays（Dual LUC）,which showed consistency with RNA-seq analysis.5.Electrophoretic mobility shift assay（EMSA）and chromatin immunoprecipitation coupled with qPCR（Ch IP-qPCR）experiment indicated that AtWRKY42 can directly bind to the promoters of many target genes.These include SAG13,SAG24,SAG103 and Yellow-Leaf-Specific gene 9/Ndr1/Hin1-Like 10（YLS9/NHL10）,Isochorismate synthase1（ICS1）,avr Pph B susceptible 3（PBS3,GH3.12）involved in SA biosynthesis,Pathogensis-Related gene1（PR1）,as well as Rboh F（Respiratory burst oxidase homolog F）implicated in ROS production.6.Genetic evidences showed that the loss of ICS1 and Rboh F genes attenuated the accumulation of SA and H₂O₂,as well as the precocious leaf senescence in AtWRKY42overexpressing lines,indicating that AtWRKY42 promotes the accumulation of SA,H₂O₂and early leaf senescence by directly regulating the expression of ICS1 and Rboh F.7.Histochemical staining and GUS activity assay of transgenic lines expressing Pro AtWRKY42-GUS showed that SA and H₂O₂ treatments repressed the AtWRKY42expression.Considering the fact that,overexpression of AtWRKY42 accelerated SA and H₂O₂ accumulation,this suggests a complex negative feedback network.Secondly,a NAC transcription factor gene BnaNAC55 in oilseed rape was found to be involved in the regulation of ROS accumulation and cell death.Its function and regulatory mechanism were analyzed through physiological and cell biological methods.The main research results are as follows.1.Analysis of BnaNAC55 protein sequence and characteristics revealed that BnaNAC55 is a member of SNAC（stress-responsive NAC）subfamily,which is phylogenetically closest to ANAC055 in Arabidopsis.Furthermore BnaNAC55 is transcriptional activator that is localized in the nucleus,and can interact with itself to form homodimers.2.Transcriptional analysis revealed that BnaNAC55 can respond to Sclerotinia sclerotiorum,cold,heat,abscisic acid（ABA）,jasmonic acid（JA）,and other biotic and abiotic stresses as well as hormone treatments.3.Overexpression of BnaNAC55 induced the accumulation of H₂O₂ and hypersensitive response（HR）-like cell death in Nicotiana benthamiana leaves,but neither the N-terminal NAC domain nor the C-terminal regulatory domain of BnaNAC55 showed such symptoms.Further physiologic measurements including relative conductivity,chlorophyll,anthocyanin,malondialdehyde（MDA）contents and DNA fragmentation also demonstrated the role of BnaNAC55 in ROS accumulation and cell death.4.Transient expression of BnaNAC55 in oilseed rape protoplasts also induced cell death.5.ANAC055,the ortholog of BnaNAC55 in Arabidopsis,had different transcriptional activity compared to BnaNAC55,and overexpression of ANAC055 could not induce ROS accumulation or cell death.6.qRT-PCR analysis showed that BnaNAC55 can regulate the expression of genes related to ROS production and scavenging,defense response and senescence.A Dual LUC assay further verified that BnaNAC55 can activate the expression of several ROS and defense related genes.Taken together,this study revealed that WRKY42 transcription factor in Arabidopsis can positively regulate leaf senescence,and seveal downstream target genes were confirmed.In addition,overexpression of AtWRKY42 promoted the early flowering of Arabidopsis to shorten its growth cycle without affecting seed yield,which is of great theoretical significance for crop breeding.Furthermore,this study analyzed the function of NAC55 gene in oilseed rape and studied its regulatory mechanism in the accumulation of ROS and cell death,which is of great theoretical value.