Transcriptome profiling of MYB-overexpressed transgenic lines provides crucial molecular insights into anthocyanin and remodel the biosynthesis regulatory network in Nicotiana tabacum

Anthocyanin is an important pigment in plants and one of the most powerful antioxidants for the food industry. In plants, anthocyanin biosynthesis is controlled by several structural genes and regulated by MYB transcription factors. However, the molecular mechanism and regulation of anthocyanin biosynthesis and the explicit role of MYB in pigmentation and stress responses have not been elucidated yet. In this study, we conducted transcriptomic profiling of MYB overexpressed transgenic lines, unveiling differentially expressed genes (DEGs) likely involved in anthocyanin biosynthesis and abiotic stress responses. Over-expression of two peanut MYB genes, Arahy.J3K16K (JK) and Arahy.CD1PG8 (C8), individually, as well as their co-expression (CJ), led to alterations in anthocyanin content in tobacco. Notably, JK over-expression line exhibited superior maintenance of growth indices, chlorophyll pigments, and anthocyanin content. Transcriptome profiling identified thousands of DEGs across JK, C8 and CJ transgenic plants. Functional annotation analysis revealed enrichment in metabolic pathways such as flavonoid biosynthesis, MAPK signaling, and hormone signaling transduction among these DEGs. Specifically, the expression of DEGs involved in the anthocyanin pathway, such as PAL, CHS, CHI, F3H, DFR, and ANR, was upregulated in JK lines, suggesting their regulatory role in pigmentation. Additionally, DEGs related to MAPK cascade, ethylene, auxin, and abscisic acid were preferentially expressed in JK lines, indicating their potential involvement in abiotic stress responses. The co-expression complex of MYB, bHLH, and WD40 also revealed the transcriptional activation model of anthocyanin biosynthesis in JK lines. In conclusion, these findings provide crucial molecular insights into MYB-induced regulation of anthocyanin biosynthesis and abiotic stress tolerance in tobacco, offering potential strategies for enhancing anthocyanin content and improving resistance.