Selection and functional identification of Dof genes expressed in response to nitrogen in Populus simonii × Populus nigra

In plants, Dof transcription factors are involved in regulating the expression of a series of genes related to N uptake and utilization. Therefore, the present study investigated how DNA-binding with one finger (Dof) genes are expressed in response to nitrogen (N) form and concentration to clarify the role of Dof genes and their functions in promoting N assimilation and utilization in poplar. The basic characteristics and expression patterns of Dof genes in poplar were analyzed by the use of bioinformatics methods. Dof genes expressed in response to N were screened, after which the related genes were cloned and transformed into Arabidopsis thaliana; the physiological indexes and the expression of related genes were subsequently determined. The function of Dof genes was then verified in Arabidopsis thaliana plants grown in the presence of different N forms and concentrations. Forty-four Dof genes were identified, most of which were expressed in the roots and young leaves, and some of the Dof genes were expressed under ammonia- and nitrate-N treatments. Three genes related to N induction were cloned, their proteins were found to localize in the nucleus, and PnDof30 was successfully transformed into Arabidopsis thaliana for functional verification. On comparing Arabidopsis thaliana with WT Arabidopsis thaliana plants, Arabidopsis thaliana plants overexpressing the Dof gene grew better under low N levels; the contents of soluble proteins and chlorophyll significantly increased, while the soluble sugar content significantly decreased. The expressions of several AMT, NRT, and GS genes were upregulated, while the expressions of several others were downregulated, and the expression of PEPC and PK genes significantly increased. In addition, the activity of PEPC, PK, GS, and NR enzymes significantly increased. The results showed that overexpression of PnDof30 significantly increased the level of carbon and N metabolism and improved the growth of transgenic Arabidopsis thaliana plants under low-N conditions. The study revealed the biological significance of poplar Dof transcription factors in N response and regulation of related downstream gene expression and provided some meaningful clues to explain the huge difference between poplar and Arabidopsis thaliana transformed by exogenous Dof gene, which could promote the comprehensive understanding of the molecular mechanism of efficient N uptake and utilization in trees.