AtMYB11/12/111 transcription factors regulate nicotine biosynthesis and enhance defense responses in tobacco

Tobacco (Nicotiana tabacum) is a globally important commercial crop, largely due to the accumulation of nicotine, a parasympathomimetic alkaloid that contributes to its economic value and ecological fitness. While the nicotine biosynthetic pathway has been well characterized, its transcriptional regulation remains incompletely understood. MYB transcription factors are established regulators of plant secondary metabolism, particularly flavonoid biosynthesis; however, their role in nicotine biosynthesis remains unexplored. Here, we demonstrate that the Arabidopsis R2R3-MYB transcription factors AtMYB11, AtMYB12, and AtMYB111 function as positive regulators of nicotine biosynthesis in tobacco for the first time. Overexpression of these MYBs led to significant upregulation of key nicotine pathway genes, including NtPMT, NtODC, NtQPT, NtMPO, NtBBL, and NtA622, resulting in increased nicotine accumulation in both seedlings and mature plants. Notably, AtMYB111 exhibited the strongest regulatory effect. Promoter analysis revealed the presence of MYB-responsive elements in NtODC and NtQPT, and both transient expression assays and yeast one-hybrid studies confirmed direct binding of AtMYB111 to these promoters. Furthermore, exogenous application of an MYB11/12/111-derived complementary peptide (cPEP) recapitulated the induction of nicotine biosynthesis, supporting a functional role of MYB-mediated regulation. Functionally, increased nicotine and flavonoid levels in MYB-overexpressing lines conferred enhanced resistance to the fungal pathogen Alternaria solani and the herbivore Helicoverpa armigera, indicating a direct link between MYB-regulated metabolism and plant defense. Collectively, this study uncovers a previously unrecognized role of these MYB transcription factors in alkaloid biosynthesis and highlights their potential for metabolic engineering of nicotine content and stress resilience in tobacco.