ATDOF5.8 protein is the upstream regulator of ANAC069 and is responsive to abiotic stress

• ATDOF5.8 can bind to DOF motifs present in the promoter of ANAC069.
• ATDOF5.8 is the up-stream regulator of ANAC069.
• A regulatory network model mediated by ANAC069 in abiotic stress is proposed.

ANAC069 encodes a plasma membrane-bound NAC protein that integrates auxin and salt signals to regulate Arabidopsis thaliana seed germination. However, the regulatory network of ANAC069 has not been revealed. Here, we report that ANAC069 is a direct target gene of the ATDOF5.8 transcription factor. There are seven DOF motifs in the promoter of ANAC069, which is highly enriched in the promoter. Yeast one-hybrid (Y1H) assays showed that a Dof protein, ATDOF5.8, binds to the DOF motifs. The interaction between ATDOF5.8 and the promoter of ANAC069 was further confirmed by transient expression assays in tobacco leaves. Chromatin Immunoprecipitation (ChIP) results suggested that the binding of ATDOF5.8 to DOF motifs in the promoter of ANAC069 occurs in vivo in Arabidopsis to regulate the expression of ANAC069. Moreover, ATDOF5.8 and ANAC069 share similar expression patterns in response to salt, drought and ABA treatment. Taken together, the results suggested a regulatory network model mediated by ANAC069, under abiotic stress conditions, ATDOF5.8 regulates the expression of ANAC069, and the activated ANAC069 binds to NAC recognition sequences or other motifs to regulate the expression of genes containing these motifs in their promoters.