Ectopic Expression of AsDof6.5 from Oat (Avena sativa L.) Enhances Low Nitrogen Tolerance in Transgenic Arabidopsis thaliana

Background: Oat (Avena sativa L.) is a vital forage and cereal crop widely cultivated on the Qinghai–Tibet Plateau, known for its outstanding adaptability to barren soils. Nitrogen (N) is a critical macronutrient, and the excessive application of N fertilizers has triggered severe ecological crises. Identifying key genes regulating adaptation to low N stress is essential for breeding N-efficient oat varieties. The DNA-binding with one finger (Dof) transcription factors play crucial roles in plant stress responses, but their precise molecular mechanisms in oat N-deficiency adaptation remain largely unknown. Results: In this study, we conducted a comprehensive genome-wide identification and unveiled 54 AsDof genes in the oat genome. Phylogenetic, synteny, and promoter analyses revealed their high evolutionary conservation and complex potential roles in stress networks. Transcriptome profiling under low N stress pinpointed AsDof6.5 as a potential candidate. Ectopic expression of AsDof6.5 in Arabidopsis thaliana significantly enhanced plant adaptation to low N. This was morphologically manifested as enhanced primary root elongation, increased root hair number, and significantly higher fresh weight under N-deprived conditions. Physiological assays further demonstrated that AsDof6.5 overexpression efficiently reduced malondialdehyde (MDA) accumulation while simultaneously preserving soluble protein (SP) levels. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays demonstrated that AsDof6.5 has the potential to physically interact with the transcription factor AsWRKY3 and the cytokinin dehydrogenase AsCKX2 in the nucleus. Furthermore, AsDof6.5 overexpression caused a significant transcriptional suppression of downstream CKX2 homologs, which likely serves to fine tune local cytokinin homeostasis, thereby sustaining root developmental plasticity and vitality under severe low N stress. Conclusions: Our findings reveal a novel protein complex transcriptional regulatory hypothetical module. This module effectively enhances adaptation to low N by integrating nutrient starvation signaling with cytokinin homeostasis, providing a theoretical basis and valuable genetic resources for breeding N-efficient crops.