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 (Nicotiana tabacum L.) remains poorly characterized. Here, we performed a genome-wide identification and comprehensive analysis of Dof genes in tobacco, revealing 75 NtDof 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 NtCDF genes respond dynamically to cold and magnesium deficiency stresses. Virus-induced gene silencing (VIGS) of two representative NtCDF genes, NtDof40 and NtDof63, revealed their opposing roles in cold tolerance: NtDof40 acts as a positive regulator, while NtDof63 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.