Dissecting The Molecular Mechanisms Of Canola NAC103 And Arabidopsis WSK1 Transcription Factors In Regulating ROS-dependent Cell Death And Low Potassium Response, Respectively

During the process of growth and development, plants are frequently affected by a variety of biotic and abiotic stresses besides their own genetic backgrounds. Plants could cope with these stresses by altering their own gene expression levels, and deploying protein modification mechanisms, in which regulation at the transcriptional level performed by transcription factors plays an extremely important role. In this study, we identified and studied a canola NAC transcription factor gene whose expression elicited reactive oxygen species(ROS) accumulation and cell death in Nicotiana benthamiana leaves. Furthermore, we preliminarily investigated the molecular mechanism of a WRKY transcription factor gene in regulating low potassium response in Arabidopsis.Firstly, we identified and characterized BnaNAC103 gene isolated from canola(Brassica napus L.) and we found that BnaNAC103 responds to multiple stresses, including cold, heat, abscisic acid(ABA), jasmonic acid(JA), salicylic acid(SA) and a necrotrophic fungal pathogen Sclerotinia sclerotiorum(S.s), which was demonstrated by quantitative reverse transcription PCR(qRT-PCR) assay. Using green fluorescence protein(GFP) gene as a reporter for fusion protein expression in tobacco leaves, we found that BnaNAC103 is exclusively located in the nucleus. Full-length BnaNAC103, but not either the N-terminal NAC domain or C-terminal regulatory domain, induces ROS accumulation and hypersensitive response(HR)-like cell death when expressed in leaves of Nicotiana benthamiana. Furthermore, BnaNAC103 expression causes obvious nuclear DNA fragmentation. Moreover, qRT-PCR analysis identified that the expression levels of multiple genes regulating ROS production and scavenging, defense response as well as senescence are significantly induced. Using a dual luciferase reporter assay system, we further confirmed that BnaNAC103 could activate the expression of a few ROS- and defense-related genes. In addition, a series of physiological, biochemical and molecular genetic assays were performed to explore the regulatory mechanism.Secondly, WRKY transcription factors are plant-specific and play important roles in many plant signaling pathways. In a previous work, our lab conducted phenotypic analysis of many Arabidopsis mutants and found that a WRKY transcription factor gene mutant wsk1(WRKY sensitive to low K+ 1)was highly sensitive to low potassium(LK) treatment. qRT-PCR analysis showed that AtWSK1 was responsive to various treatments including cold, ABA, low phosphorous( LP), etc. Phenotypic analysis confirmed that wsk1 was hypersensitive to low potassium at both germination and post-germination level. Microarray profiling of wild-type(WT) and wsk1 mutant under LK treatment was performed and a few hundren differentially expressed genes(DEGs) were identified, part of which were validated through qRT-PCR. Furthermore, we used qRT-PCR to analyze the transcriptional level of a bunch of K+ channel and transporter genes between mutant and WT under both normal and LK treatments. Yeast one-hybrid was also used to test the binding of WSK1 transcription factor to its putative target genes. Moreover, yeast two-hybrid assay demonstrates that a mitogen-activated protein kinase(MAPKs/MPKs), MPKy interacted with WSK1. Besides, MPKx interacted with the cytosolic domains of some K+ channel proteins. Furthermore, selected interactions were verified in Nicotiana benthamiana by bimolecular fluorescence complementation(BiFC) assay. To our knowledge, this is the first report that WRKY transcription factor regulates plant reponse to low potassium and that, MPK-WRKY signaling pathway are likely involved in this process. Our work has laid a solid foundation for further exploring the molecular mechanisms of MPK-WRKY signaling pathway in regulating low potassium response.