Identification and characterization of sub-group 4 R2R3-MYB members and their functions in phenylpropanoid and flavonoid regulation in tobacco

Sub-group 4 R2R3-type MYB transcription factors (TFs) are involved in repression of the phenylpropanoid pathway. In this study, we identified and characterized three MYB transcriptional repressors in common tobacco, named NtMYB1, NtMYB2, and NtMYB4, which belong to sub-group 4 members. Tissue expression properties and specificity of stress-induced expression indicated that the NtMYB1, NtMYB2, and NtMYB4 may perform different functions in tobacco. Overexpression of NtMYB1, NtMYB2, and NtMYB4 reduced phenylpropanoids and flaonoid compounds, such as caffeic acid, chlorogenic acid, neochlorogenic acid, and rutin, respectively. Meanwhile, the expression of the most structural genes involved in phenylpropanoids and flavonoids were reduced in the overexpression transgenic lines. In addition, results based on co-expression and expression analyses revealed possible interactions between NtMYB1, NtMYB2, and NtMYB4 and multiple bHLH TFs. Our results showed that the three sub-group 4 R2R3 members exhibited different expression pattern in various tissues and under abiotic stress conditions. Meanwhile, we hypothesized that the three of them involved in the inhibition of phenylpropanoid and flavonoid biosynthesis might be interaction with bHLH TFs.