NtMBD1 regulates shoot branching regulation and evolutionary differentiation in tobacco (Nicotiana tabacum L.)

Branching regulation and biomass accumulation are key determinants of tobacco productivity and bioenergy potential, yet the molecular mechanisms underlying branching control in tobacco remain poorly understood. Here, we characterize the ntmbd1 mutant derived from EMS mutagenesis, which exhibits excessive basal branching and altered plant architecture. SSR mapping and whole-genome sequencing identify a nonsense mutation in ntmbd1 leading to truncation of the encoded P450 protein. NtMBD1 is predominantly expressed in the shoot apical meristem, flower, and root, where its localization to the endoplasmic reticulum suggests a role in ER-associated regulatory processes. Phylogenetic analysis reveals that NtMBD1 in cultivated tobacco originates from its ancestral species Nicotiana sylvestris, while its homolog from N. tomentosiformis is likely lost during evolution. Functional validation shows that NtMBD1 is required for axillary bud suppression, as CRISPR disruption in N. tabacum and N. benthamiana phenocopies the branching defect, while heterologous complementation of the Arabidopsis max1 mutant confirms its conserved function; moreover, complementation of the ntmbd1 mutant restores normal architecture and biomass traits. Collectively, these findings reveal NtMBD1 as a key regulator of branching and plant architecture, providing new insights into its molecular and evolutionary roles and offering potential applications in crop improvement and sustainable bioenergy development.