Graduate student; Graduate research assistant University of Kentucky Lexington, Kentucky
Body of Abstract: Nicotine is an essential specialized metabolite that tobacco produces mainly for resisting biotic stresses. The transcriptional regulation of nicotine biosynthesis has been extensively studied; however, the post-translational regulation mechanisms involved in the pathway remain unclear. The mitogen-activated protein kinase (MAPK) cascades comprise at least 3 kinases, including MAP3K, MAP2K, and MAPK. The upstream kinase (MAP3K) phosphorylates the downstream kinase (MAP2K), then phosphorylates MAPK, which phosphorylates multiple protein substrates, such as transcription factors, resulting in the modification of protein activities. MAPK cascades are well-known stress-responsive regulators; however, how they are involved in specialized metabolism is poorly understood.
Our previous work has demonstrated that the tobacco MAPK, NtMPK4, is a positive regulator of nicotine biosynthesis. However, the upstream MAP3K and MAP2K in the signaling cascade have not been identified.
Here, we identified NtMEKK1b and NtMKK2s as the upstream kinases of NtMPK4 in the cascade. Protein-protein interaction assays demonstrated that NtMKK2a and NtMKK2b interact with NtMPK4 and that the NtMEKK1b interacts with NtMKK2a in yeast and plant cells. NtMKK2a phosphorylates NtMPK4in vivo. Promoter transactivation assays demonstrated that NtMEKK1b and NtMKK2s enhance the activity of a nicotine biosynthesis-related transcription factor, NtERF221. The overexpression of NtMEKK1b or NtMKK2a in tobacco hairy roots increased the expression of nicotine biosynthetic genes and nicotine contents. Knocking down NtMEKK1b in transgenic plants resulted in reduced expression of nicotine biosynthetic genes and nicotine contents. Knocking down NtMKK2s in tobacco hairy roots decreased the nicotine content and pathway gene expression.
In summary, our findings indicated that the NtMEKK1b-NtMKK2-NtMPK4 cascade plays a positive role in the post-translational regulation of nicotine biosynthesis. This work helps decipher the post-translational regulatory mechanisms of specialized metabolism in plants.