Evolutionary analysis of the Dof family in plants and identification of its members in tobacco

DNA-binding with One Finger (Dof) transcription factors are plant-specific regulators involved in diverse biological processes, including growth, development, and stress responses. However, the Dof family in tobacco (<i>Nicotiana tabacum</i> L.) remains poorly characterized. Here, we performed a genome-wide identification and comprehensive analysis of <i>Dof</i> genes in tobacco, revealing 75 <i>NtDof</i> members. Through phylogenetic reconstruction, gene structure analysis, and cis-regulatory element prediction, we classified these genes into seven subfamilies and found that the CDF clade exhibits conserved exon-intron structures, unique motif compositions, and enrichment of stress- and hormone-responsive cis-elements. Expression profiling showed that most <i>NtCDF</i> genes respond dynamically to cold and magnesium deficiency stresses. Virus-induced gene silencing (VIGS) of two representative <i>NtCDF</i> genes, <i>NtDof40</i> and <i>NtDof63</i>, revealed their opposing roles in cold tolerance: <i>NtDof40</i> acts as a positive regulator, while <i>NtDof63</i> functions as a negative regulator. Our findings provide a comprehensive evolutionary framework for the Dof family in tobacco and highlight the functional divergence of CDF subfamily members in abiotic stress adaptation, offering potential targets for stress-resistance breeding.