The Regulatory Mechanisms Of Four NAC Transcription Factors In Modulating ABA-induced Postharvest Leaf Senescence Of Chinese Flowering Cabbage

Chinese flowering cabbage(Brassica rapa ssp.Parachinensis)is one of an important leafy vegetable in southern China with high economically value.This leafy vegetable is known and popular to consumers as it is a good source of minerals,vitamins,and health-promoting compounds.However,leaves of Chinese flowering cabbage are easy to senesce and become yellow after harvest,leading to short shelf-life and substantial economical loss.Thus,understanding the regulatory mechanisms of leaf senescence is critical for improving the technology to maintain quality and prevent postharvest loss of this important leafy vegetable.Phytohormones are important regulators of leaf senescence in plants,moreover,diverse phytohormones regulate leaf senescence synergistically or antagonistically through combination of complicated networks.In general,abscisic acid(ABA),ethylene and jasmonic acid(JA)are the major plant hormones promoting leaf senescence,whereas others like cytokinins(CTK)and gibberellins(GA)have been shown to delay senescence.Transcription factors play an important role in the crosstalks of phytohormones.Plant-specific transcription factors NACs are well-documented to be crucial regulators of leaf senescence by affecting ABA biosynthesis.Our previous studies showed that exogenous application of GA suppressed Chinese flowering cabbage leaf senescence,while ABA treatment accelerated leaf senescence,however,it remains largely unknown whether crosstalk between ABA and GA exists during Chinese flowering cabbage leaf senescence,as well as the regulatory mechanisms.Therefore,in the present work,changes in endogenous ABA and GA levels and expression levels of genes associated with ABA and GA biosynthesis and catabolism,during ABA-induced leaf senescence of Chinese flowering cabbage were investigated.Differential expressed senescence-related transcription factors were identified,among which NAC transcription factors were mainly focused.Direct targets and their regulatory effects of four NAC transcription factors were confirmed by electrophoretic mobility shift assay(EMSA),chromatin immunoprecipitation(Ch IP)-q PCR assay and dual luciferase reporter assay(DLR).Furthermore,biological function of one NAC transcription factor was verified through transgenetic approach in Arabidopsis.The main results are listed as follows:1.Exogenous application of ABA accelerated leaf senescence of Chinese flowering cabbage,evidenced by the data that ABA treatment led to the stronger reduction in total chlorophyll content and the maximum quantum yield(Fv/Fm),while significant induction in ROS accumulation and the expression of several senescence-marker gene(SAG)Br SAG12,chlorophyll catabolic genes(CCGs)Br NYE1,Br NYC1,Br NOL1,Br PPH1,Br PAO and Br RCCR,and genes encoding ROS-producing enzymes respiratory burst oxidase homologues(RBOHs)Br Rboh B and Br Rboh C.Levels of endogenous ABA increased,while GA decreased during leaf senescence.Obviously enhanced accumulations of endogenous ABA,and reduction of GA content were also found after ABA treatment,as ABA biosynthetic genes Br NCED3 and Br ABA2,and GA catabolic gene Br GA2ox1 were up-regulated,but ABA catabolic gene Br CYP707A3,and GA biosynthetic genes Br KAO2 and Br GA20ox2,were down-regulated by ABA treatment.These results suggest that ABA accelerates leaf senescence of Chinese flowering cabbage,possibly by enhancing ROS production,ABA accumulation,chlorophyll and GA degradation,and inhibiting ABA catabolism and GA biosynthesis,leading to increased ABA levels and decreased GA levels.2.A transcriptome database associated with ABA-induced leaf senescence of Chinese flowering cabbage was established using RNA-seq.12,361 differentially expressed genes including 6,316 up-regulated genes and 6,045 down-regulated genes were identified during leaf senescence,using a threshold false discovery rate(FDR)of < 0.05 and an absolute value of log2 Ratio > 1.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis showed that the differentially expressed genes were significantly enriched in plant hormones biosynthesis and metabolism,and transduction of hormone signals.898 differentially expressed transcription factors were further identified,notably ERF,NAC,b HLH,WRKY and MYB.Moreover,four NAC transcription factors that were obviously induced by ABA,were focused.These four NAC transcription factors exhibited the highest identity with Arabidopsis ANAC041,ANAC055,ANAC087 and ANAC092;so they were designated as Br NAC041,Br NAC055,Br NAC087 and Br NAC092,respectively.Subcellular localization assay showed that Br NAC041,Br NAC055,Br NAC087 and Br NAC092 were localized in the nucleus.Transcriptional ability analysis in yeast and tobacco leaves indicated that Br NAC055,Br NAC087 and Br NAC092 possessed trans-activation ability,while Br NAC041 showed trans-repression activity.3.Direct targets and their regulatory effects of Br NAC041,Br NAC055,Br NAC087 and Br NAC092 were confirmed by EMSA,Ch IP-q PCR and DLR assays.Br NAC055 directly activated the transcriptions of Br Rboh B,Br Rboh C,Br NYC1 and Br NYE1,thereby accelerating leaf senescence in Chinese flowering cabbage.Moreover,transient overexpression of Br NAC055 in tobacco leaves made an increased ROS level and accelerated chlorophyll degradation via the up-regulation of Nb Rboh A and Nb SGR1,resulting in the promoted leaf senescence.Polyclonal anti-Br NAC041,-Br NAC087 and-Br NAC092 antibodies were prepared,and protein levels of Br NAC041,Br NAC087 and Br NAC092 were also increased during leaf senescence,and remarkably induced by exogenous ABA treatment.Further experiments clearly revealed that Br NAC087 and Br NAC092 directly targeted the promoters of Br SAG12,Br NYE1,Br NYC1,Br NOL1,Br PPH1,Br PAO,Br RCCR,Br NCED3,Br ABA2 and Br GA2ox1 by recognizing the NACRS,and activated their transcriptions.In addition,Br NAC041 repressed the transcription of Br KAO2,Br GA20ox2 and Br CYP707A3.Collectively,these findings demonstrate that Br NAC041,Br NAC087 and Br NAC092 are involved in ABA-accelerated leaf senescence of Chinese flowering cabbage via direct regulation of chlorophyll degradation,ROS accumulation,endogenous ABA and GA biosynthesis and catabolism.4.Biological function of Br NAC092 was verified by transiently overexpressed in tobacco leaves and stably over-expressed in Arabidopsis,respectively.Transient overexpression of Br NAC092 in tobacco leaves made an increased ROS level and accelerated chlorophyll degradation via the up-regulation of Nb Rboh A,Nb SGR1,ABA biosynthetic and GA catabolic genes,leading to the accelarated leaf senescence.The ectopic overexpression of Br NAC092 in Arabidopsis also confirmed that Br NAC092 functioned in ABA-induced leaf senescence by modulating chlorophyll degradation and ROS accumulation,as well as ABA and GA biosynthesis and catabolism.