Functional Characterization Of One NAC And One BHLH Transcription Factors Regulating Leaf Senescence And Development,Respectively In Arabidopsis Thaliana

Plant leaves are crucial organs for photosynthesis and sensing changes in the environment,and play an important role in plant growth and development.The whole life process of leaves can be divided into four stages: the forming of leaf primordia,polarity establishment,growth and senescence,which need to rely on the fine regulation of transcription factors.The functions of AtNACx and AtbHLH? transcription factors in leaf senescence and leaf morphogenesis were studied in this study.Firstly,we found that Arabidopsis thaliana AtNACx was involved in the regulation of leaf senescence.The expression of AtNACx is induced by chilling,heat,salt stress and ABA treatments.Subcellular localization assay showed that AtNACx was located at the endoplasmic reticulum,whereas AtNACx-?TM in the nucleus.qRT-PCR analysis showed that the expression of AtNACx decreased gradually with age-dependent and dark-induced leaf senescence,and only increased at the later stage of age-dependent leaf senescence.Through the phenotypic analysis of gain-of-function mutant of AtNACx,we found that nacx showed delayed senescence and higher chlorophyll content compared with wild-type in age-dependent and dark-induced leaf senescence.There was no significant difference between AtNACx-RNAi and WT lines in age-dependent senescence,but AtNACx-RNAi show early senescence and had lower chlorophyll content than WT under dark-induced condition.In Arabidopsis thaliana seedlings,induced expression of AtNACx-?TM promoted accumulation of ROS and DNA fragmentation.In the phenotypic analysis,it was found that induced expression of AtNACx-?TM could rapidly induce leaf senescence and accumulation of ROS in Arabidopsis thaliana.AtNACx-?TM was able to activate the expression of AtRBOHD,AtRBOHF,AtZAT12,AtAOX1 a,AtSAG12,AtSAG29,AtNCED3 and AtDREB2 B genes by qRT-PCR and dual LUC analyses.Electrophoretic mobility shift assay(EMSA)indicated that AtNACx can bind directly to the promoter regions of AtSAG12 and AtSAG29.At the same time,it was found that transient expression of AtNACx-?TM in N.benthamiana leaves could lead to the accumulation of ROS and hypersensitive response-like cell death,possibly through modulating the expression of ROS-and defense-related genes.Secondly,a previous study in our lab found that the leaves of overexpression lines of AtbHLH? were serrated,so I further studied the mechanism of AtbHLH? in the process.Through subcellular localization and transcriptional activity analysis,it was found that AtbHLH? transcription factor was localized in the nucleus and had transcriptional activation activity.Further,it was found that AtbHLH? could form homodimer and heterodimer with itself and paralogous AtbHLH?,respectively,and the transcriptional activation activity of AtbHLH? was inhibited by AtbHLH?.In the phenotypic analysis of AtbHLH? mutants and transgenic lines,we found that a more abnormal leaf phenotype appeared when AtbHLH? was overexpressed in the bhlh?-2 mutant.For instance,some of these lines were even fibrous.Further studies showed that AtbHLH? can interact with AtJLO,AtLBD18,AtLBD25 and AtLOB belonging to LBD(lateral organ boundaries)family in Arabidopsis,and AtJLO,AtLBD18 and AtLOB could inhibit the transcriptional activation activity of AtbHLH?.In addition,biomolecular fluorescence complementation(BiFC)experiments found that AtbHLH? can also interact with AtLBD18,At LBD25,AtLOB,respectively.More interestingly,it is also found that AtbHLH? interacts with AtAS1(MYB91)and AtAS2 transcription factors,which act in the establishment of leaf kyphotic polarity,and AtbHLH? can regulate the expression of AtBP1.The above results demonstrated that AtbHLH? was involved in leaf morphology probably through interacting with AtJLO,AtLOB and AtAS1,AtAS2,respectively.