## 2 Results and Discussion
### Binding of ANAC074 to NRS1, NRS2 and MybSt1 Actually Occurs in Plants  
To determine whether the binding of ANAC074 to NRS1, NRS2 and MybSt1 actually occurs in plants, ChIP analysis was performed using _Arabidopsis thaliana_ plants overexpressing a ANAC074-GFP fusion gene. The three truncated promoter regions of each putative target gene (_AT2G21320_, _AT2G31880_, _AT3G13470_, _AT4G17500_, _AT1G74930_, or _AT4G11280_) with NRS1, NRS2 or MybSt1 element were examined. The results showed that the truncated promoters of _AT2G21320_, _AT2G31880_ and _AT3G13470_ all can be amplified from chromatin immunoprecipitated with anti-GFP antibody, but not from chromatin immunoprecipitated with HA antibody (Figure 4). These results suggested that ANAC074 binds to the promoter of _AT2G21320_ (containing NRS1 in promoter and no NRS2, MybSt1, and NACRS) (Figure 4A), the promoter of _AT2G31880_ (containing NRS2 in promoter and no NRS1, MybSt1, and NACRS) (Figure 4B), and the promoter of _AT3G13470_ (containing MybSt1 in promoter and no NRS1, NRS2, and NACRS) (Figure 4C). Unfortunately, we did not detect that the promoters of _AT4G17500_, _AT1G74930_, or _AT4G11280_ could be bound by ANAC074 in the ChIP experiment. The truncated promoters of _AT4G17500_, _AT1G74930_, and _AT4G11280_ could be amplified from input; however, the chromatin DNA of 355::ANAC074::GFP transgenic plants immunoprecipitated with GFP antibody (ChIP+) failed to amplify the corresponding PCR products (data not shown). Interestingly, there are regions where the binding site is present but no PCR product amplification is detected (Figure 4A-C). This phenomenon is also present in the study of [PERSON] et al. [(34)]. They examined a total of six areas containing target components, and the results showed that MdCIbHLH1 only bound to the _MdCBF2_ promoter in three regions [(34)]. It is likely that different DNA fragments containing assumed binding sites compete when binding to the same transcriptional factor. Additionally, it is also possible that a certain amount of DNA was lost in the process of DNA purification after uncrosslinking. Accumulating evidence indicates that NAC gene family members are involved in regulating ethylene signals [(35; 36)]. MdNAC047 directly binds to the _MdERF3_ (ETHYLENE RESPONSE FACTOR) promoter and activates its transcription. MdNAC047 regulates ethylene production at least partially in an MdERF3-dependent pathway [(37)]. SNAC4 and SNAC9 could positively regulate the tomato fruit ripening process by functioning upstream of ethylene synthesis genes including _LeACS2_ and _LeACS4_[(38)]. It is worth mentioning that _ATERF-1_ (_AT4G17500_), _ORA47_ (_AT1G74930_), _ACS6_ (_AT4G11280_), _BBX18_ (_AT2G21320_), _SOBIR1_ (_AT2G31880_), and _CPNB2_ (_AT3G13470_) as well as _ANAC074_ all can respond to ethylene (see Affymetrix Arabidopsis ATH1 Genome Array, GSE537), which suggests they may be involved in the ethylene signaling pathway. ATERF-1 (AT4G17500) and ORA47 (AT1G74930) both belong to the ERF/AP2 transcription factor family, which plays a crucial role in the ethylene signaling pathway [(39; 40)]. ATERF-1 encodes a member of the ERF (ethylene response factor) subfamily B-3, and ORA47 encodes a member of the DREB subfamily A-5. ACS6 is important to the SA potentiated induction of ethylene biosynthesis [(41)]. In Y1H and transient expression assays we found that ANAC074 can bind to the promoters of _ATERF-1_, _ORA47_, and _ACS6_, demonstrating that ANAC074 may be as a regulator functioning upstream of ethylene synthesis genes. However, we did not detect the interaction between ANAC074 and the promoters of these three genes using the ChIP experiment in 3-week-old seedlings under normal growth condition. On the one hand, there may be functional redundancy between ANAC074 and other NACs under normal growth conditions in _Arabidopsis_. It is possible that the promoters of _ATERF-1_, _ORA47_, and _ACS6_ are preferentially identified by ANAC069 or other NACs rather than ANAC074 when _Arabidopsis_ grows normally.