Transcription Factor ANAC074 Binds to NRS1, NRS2, or MybSt1 Element in Addition to the NACRS to Regulate Gene Expression  
[PERSON]  
1 Key Laboratory of Modern Agricultural Cultivation and Crop Germplasm Improvement of Heilongjiang Province, College of Agriculture, Heilongjiang Bayi Agricultural University, 5 Xinfeng Road, Daqing 163319, China; [EMAIL_ADDRESS] (L.H.); [EMAIL_ADDRESS] (J.X.)  
[PERSON]  
1 Key Laboratory of Modern Agricultural Cultivation and Crop Germplasm Improvement of Heilongjiang Province, College of Agriculture, Heilongjiang Bayi Agricultural University, 5 Xinfeng Road, Daqing 163319, China; [EMAIL_ADDRESS] (L.H.); [EMAIL_ADDRESS] (J.X.)  
[PERSON]  
2 State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, 26 Hexing Road, Harbin 150040, China  
2 Correspondence: [EMAIL_ADDRESS] (Y.W.); [EMAIL_ADDRESS] (K.Y.); Tel.: +86-15134564509 (Y.W.); +86-13836961072 (K.Y.)  
2 These authors contributed equally to this work.  
[PERSON]  
1 Key Laboratory of Modern Agricultural Cultivation and Crop Germplasm Improvement of Heilongjiang Province, College of Agriculture, Heilongjiang Bayi Agricultural University, 5 Xinfeng Road, Daqing 163319, China; linlinhe65@sina.  
The diverse functions of NAC TFs possibly depends on their combination with the relevant cis-acting elements existing in the promoters of their target genes. Therefore, target element identification for NAC TFs is a prerequisite for parsing the action of NAC family members in the plant. Accumulated studies have shown that NAC transcription factors mainly bind to the classic NAC recognition sequence (NACRS) with discontinuous core sequence of CATGTG and CACG in promoters to regulate the expression of target genes. In _Arabidopsis_, ANAAC019, ANAAC055, and ANAAC072 bind to a NACRS in the promoter of the early responsive to dehydration stress 1 (_ERD1_) gene, which encodes a ClpA (ATP binding subunit of the caseinolytic ATP-dependent protease) homologous protein [19]. ANAAC096 activates transcription via recognition of the NACRS sequences within the _RD29A_ promoter [20]. SNAC1 from rice can bind to the similar NACRS in the promoter of the rice _ERD1_ (early responsive to drought 1) gene [13]. In addition to discontinuous motif, NACs also bind to the continuous core sequence. For instance, both ANAAC019 and ANAAC092 from _Arabidopsis_ can bind to the TTNCGT[G/A] sequence [21]. _Arabidopsis_ NAC1 binds to a 21-bp DNA fragment containing an as-1 element (TGACG) [6]. ANAAC069 binds to C[A/G]CG[T/G] sequence to regulate the expression of stress related genes under salt and osmotic stress conditions [22]. Combining these results, it can be suggested that diverse NAC proteins can bind cis-acting elements with similar core sequences \"CACG\" or \"CGT[G/A]\".  
To date, although the functions of NACs have been studied extensively, there are still many grey areas that need to be further studied in the NAC-mediated gene regulatory networks. There has been little work on new target element identification for NAC TFs. Therefore, we were convinced that it was worthwhile pursuing the challenge to identify more target elements of NACs.  
In the present study, we identified three new interactions between NACs and DNA sequences in _Arabidopsis_ using the TF-centered Y1H system [23; 24], including two novel motifs, NRS1 and NRS2, and an MybSt1 that previously was found to only bind by Myb transcription factors. Further analysis showed that ANAC074 could bind the promoters of ethylene and stress responsive genes via the NRS1, NRS2, or MybSt1 element. These results suggested that NACs recognize more motifs than we previously knew and these motifs may play roles in expression regulation mediated by NACs. The identified motifs recognized by NACs will facilitate understanding of the function of NACs in depth.