Singh, N.; Dwivedi, S.; Singh, D.; Pathak, P. K.; Trivedi, P. K.

Anthocyanins are secondary metabolites synthesized through the phenylpropanoid pathway. They attract pollinators, possess antioxidant properties that scavenge free radicals during environmental stress, and provide protection against various stress conditions. Lignin, another secondary metabolite, plays crucial roles in providing mechanical support, facilitating water and solute transport, and protecting against pathogens. MYB transcription factors, particularly R2R3 MYBs, are key regulators of secondary metabolism, especially within the phenylpropanoid pathway. These factors act as both activators and repressors. The N-terminal region of R2R3-MYB repressors contains a conserved bHLH-binding domain, while the C-terminal domain is divergent and includes an EAR repressor domain. R2R3-MYB proteins notably target sequences such as the MYB-response element and AC elements. In this study, we identified and characterized the Nicotiana MYB transcription factor, NtMYB308, and explored its regulatory function in anthocyanin and lignin biosynthesis in tobacco. Our Virus Induced Gene Silencing (VIGS) and Protein-DNA interaction studies suggest that NtMYB308 is a negative regulator of anthocyanin and lignin biosynthesis by binding to the promoters of genes involved in these pathways. To validate our findings, we developed CRISPR/Cas9-based knockout mutant plants of tobacco, NtMYB308CR, which showed up-regulation of anthocyanin and lignin biosynthesis. Conversely, NtMYB308 overexpression (NtMYB308OX) plants exhibited the opposite effect. Enhanced anthocyanin and lignin levels in NtMYB308CR plants provided tolerance against the fungus Alternaria solani, while NtMYB308OX lines were susceptible. This study advances our understanding of the regulatory mechanisms governing anthocyanin and lignin biosynthesis and their role in biotic stress in tobacco.