Transcription Factor ANAC074 Binds to NRS1, NRS2, or MybSt1 Element in Addition to the NACRS to Regulate Gene Expression

NAC (NAM, ATAF1/2, and CUC2) transcription factors play important roles in many biological processes, and mainly bind to the NACRS with core sequences “CACG” or “CATGTG” to regulate gene expression. However, whether NAC proteins can bind to other motifs without these core sequences remains unknown. In this study, we employed a Transcription Factor-Centered Yeast one Hybrid (TF-Centered Y1H) screen to study the motifs recognized by ANAC074. In addition to the NACRS core cis-element, we identified that ANAC074 could bind to MybSt1, NRS1, and NRS2. Y1H and GUS assays showed that ANAC074 could bind the promoters of ethylene responsive genes and stress responsive genes via the NRS1, NRS2, or MybSt1 element. ChIP study further confirmed that the bindings of ANAC074 to MybSt1, NRS1, and NRS2 actually occurred in Arabidopsis. Furthermore, ten NAC proteins from different NAC subfamilies in Arabidopsis thaliana were selected and confirmed to bind to the MybSt1, NRS1, and NRS2 motifs, indicating that they are recognized commonly by NACs. These findings will help us to further reveal the functions of NAC proteins.