Isolation and molecular characterization of NtMYB4a, a putative transcription activation factor involved in...

The MYB transcription factors are involved in the regulation of plant secondary metabolism, cell development and morphogenesis, and stress response. Here, a full-length, 816-bp NtMYB4a cDNA, which encodes a protein comprising 271 amino acids, was isolated from tobacco leaves. Phylogenetic analysis revealed that NtMYB4a is most similar to Nicotiana. attenuata MYB4, followed by Eriobotrya japonica MYB4, and NtMYB4a clustered with transcriptional activators rather than repressors. Subcellular localization assays showed that NtMYB4 localized in the nucleus, membrane, and cytoplasm. Expression analyses revealed differential expression of NtMYB4a among different tissues and organs and between different developmental stages, with most expression occurring in the stems and leaves during the full-bloom stage. Moreover, NtMYB4a expression was induced by cold, NaCl, PEG, abscisic acid, methyl jasmonate, and dark stressors, and the expression patterns and maximum expression levels varied with the type of stress. Overexpression of NtMYB4a upregulated NtPAL, Nt4CL, NtCHS, NtCHI, NtF3H, NtDFR, NtANS, and NtUFGT, which resulted in increased anthocyanin content in the tobacco corolla and darker colors. However, CRISPR/Cas9-mediated knockout of NtMYB4a downregulated NtPAL, NtC4H, Nt4CL, NtCHS, NtCHI, NtF3H, NtANS, and NtUFGT, which resulted in reduced anthocyanin content, and lighter corolla colors. These results indicated that NtMYB4a positively regulates anthocyanin biosynthesis and is involved in abiotic stress responses in tobacco plants.