Author ORCID Identifier
https://orcid.org/0000-0002-7658-7368
Date Available
11-5-2027
Year of Publication
2025
Document Type
Doctoral Dissertation
Degree Name
Doctor of Philosophy (PhD)
College
Agriculture, Food and Environment
Department/School/Program
Plant and Soil Sciences
Faculty
Ling Yuan
Faculty
Authur G. Hunt
Abstract
In tobacco (Nicotiana tabacum), nicotine is a key specialized alkaloid involved in defense and is also a major commercial concern due to its addictive nature. However, most genetic approaches targeting transcription factors or structural enzymes involved in nicotine biosynthesis either severely impair plant growth or fail to reduce nicotine levels sufficiently, highlighting the need for alternative regulatory mechanisms. Previous studies identified NtMPK4, a mitogen-activated protein (MAP) kinase, as a positive regulator of nicotine biosynthesis, underscoring the importance of post-translational regulation. MAP kinases function as cascades that are composed of three core kinases: a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and a MAP kinase (MAPK).
Our overarching hypothesis was that a canonical MAPK module regulates nicotine biosynthesis in response to environmental biotic signals such as phytohormone jasmonate (JA) and the bacterial elicitor flg22. The components of the NtMPK4 cascade and its regulatory function in nicotine biosynthesis remained unclear. While previous studies have highlighted the transcriptional regulation of nicotine pathway genes by JA-responsive transcription factors, the post-translational mechanisms that fine-tune these responses remain poorly understood.
This dissertation elucidates the upstream and downstream components of the NtMPK4 cascade. Through phylogenetic, co-expression, and spatial expression analyses, we identified NtMPKK2a as an upstream kinase of NtMPK4. Protein-protein interaction assays (yeast two-hybrid and luciferase complementation) and phos-tag analysis confirmed that activated NtMPKK2a phosphorylates NtMPK4. Overexpression of NtMAPKK2a enhanced the transactivation activity of NtERF221 on the promoter of NtPMT, a key nicotine biosynthetic gene, and increased nicotine levels in transgenic tobacco hairy roots. RNAi silencing of NtMPKK2a reduced nicotine content. Similarly, we identified NtMEKK1b as the upstream MAPKKK of NtMPKK2a. Overexpression of NtMEKK1b enhanced the transactivation of the NtPMT promoter and increased nicotine levels in transgenic tobacco hairy roots, whereas silencing NtMEKK1b suppressed it.
We aimed to identify the substrates of NtMPK4. We identified a WRKY transcription factor and co-activation factor NtVQ22, as substrates of NtMPK4. NtVQ22 interacts with NtMPK4 and the C-terminal of NtWRKY1. NtWRKY1 induced the transactivation of NtPMT and NtQPT promoters in leaf-based transactivation assays. Transcriptome analysis of tobacco hairy roots overexpressing NtWRKY1 revealed that the expression of nicotine biosynthetic pathway genes was upregulated in the NtWRKY1-OE lines compared to the control, and the nicotine contents increased to 155% to 193% compared to control, respectively. Apart from the VQ-WRKY complex, we also tested several nicotine-related transcription factors. NtMPK4 physically interacts with the AP2/ERF factor NtERF221 and the bHLH factor NtMYC2, and enhances their transcriptional activity, leading to upregulation of nicotine biosynthetic genes. These results indicated that NtMPK4 regulate nicotine biosynthesis via the NtVQ22-NtWRKY1 complex as well as the NtMYC2-NtERF221 module. These findings suggest that NtMPK4 interacts with multiple substrates and orchestrates diverse downstream pathways to regulate nicotine biosynthesis.
One of the most notable aspects of this study is the demonstration that NtMEKK1b is activated by multiple external stimuli. We showed that both JA and flg22 treatments enhanced NtMEKK1b phosphorylation in tobacco leaves. Overall, this study demonstrates that the NtMEKK1b-NtMPKK2a-NtMPK4 cascade is sequentially activated in response the external stimuli, leading to the activation of NtVQ22-NtWRKY1 complex and the NtMYC2-NtERF221 module, which in turn enhance the expression of nicotine biosynthetic genes. The dissertation elucidates a novel post-translational regulatory pathway controlling nicotine biosynthesis, providing an alternative to traditional transcriptional regulation. These findings offer valuable insights into future research on specialized metabolism and strategies for nicotine control.
Digital Object Identifier (DOI)
https://doi.org/10.13023/etd.2025.454
Recommended Citation
Zhou, Yan, "POST-TRANSLATIONAL REGULATION OF ALKALOID BIOSYNTHESIS IN TOBACCO (Nicotiana tabacum)" (2025). Theses and Dissertations--Plant and Soil Sciences. 196.
https://uknowledge.uky.edu/pss_etds/196
