## 2 Results and Discussion
### Binding of ANAC074 to NRS1, NRS2 and MybSt1 Actually Occurs in Plants
On the other hand, the interaction between ANAC074 and the promoter of _ATERF-1_, _ORA47_, or _ACS6_ in _Arabidopsis_ may be determined by the growth period and the environmental conditions of the plants. Perhaps this interaction occurs only during specific growth stage or under certain adverse conditions. Further study was required to reveal when and under what conditions ANAC074 binding to the promoters of _ATERF-1_, _ORA47_, and _ACS6_ occurs in _Arabidopsis thaliana_. Figure 3: ANAC074 can bind the promoter of _AT4G17500, AT1G74930,_ and _AT4G11280_ via the NRS1, NRS2 or MybSt1 element. (**A**) Schematic diagram of the truncated promoters containing NRS1, NRS2, or MybSt1, without NRS1, NRS2, or MybSt1, or with the mutated NRS1, NRS2, or MybSt1 used in Y1H analysis. NRS1p+, NRS2p+, MYBp+: the truncated promoter with NRS1, NRS2, or MybSt1. NRS1p-, NRS2p-, MYBp-: the truncated promoter without NRS1, NRS2, or MybSt1. NRS1 pm, NRS2 pm, MYBpm: the truncated promoter with the completely mutated NRS1, NRS2, or MybSt1. Black shading indicates NRS1, NRS2, or MybSt1 motif. Dark grey shading indicates completely mutated NRS1, NRS2, or MybSt1 motif. (**B**) Analyses of the binding of ANAC074 to the truncated promoters containing NRS1, NRS2, or MybSt1 motif using Y1H. Positive transformants were further identified by spotting yeast cells with serial dilutions (1/1, 1/10, 1/100, 1/1000) onto medium of SD/-His/-Leu/-Trp with 60 mM 3-AT. The transformants grown at SD/-Leu/-Trp were as positive controls for transformant growth. Positive control: pGADT7-p53 interacting with p53 HIS2; Negative control: pGADT7-ANAC074 interacting with p53 HIS2. (**C**) Schematic diagram of the effector and reporter constructs used in vivo transient expression assays. (**D**) Analyses of the bindings of ANAC074 to the truncated promoters containing NRS1, NRS2, or MybSt1 motif used in the coexpression experiments. The different combinations between effector and reporter are shown in the left panel. The empty pCAMBIA1301 was as positive control. Co-transformations of empty pROKII with pCAM-NRS1p+, pCAM-NRS2p+ or pCAM-MYBp+ were as negative controls. Data represent mean values of three independent experiments, and bars indicate standard deviation (SD). Asterisks indicate a significant difference compared with NRS1p-, NRS2p-, or MYBp- (Student’s test; \({}^{*}p<0.05\)). Some NAC transcriptional factors are involved in biotic or abiotic stress response through the ethylene signaling pathway. For example, _Arabidopsis_ transcription factor NAC2 is involved in the salt stress response through the ethylene signaling pathway [36]. [PERSON] et al. found a novel \"MdNAC047-_MdERF3_-ethylene-salt tolerance\" regulatory pathway, which provide new insight into the link between ethylene and salt stress [37]. In this work, ANAC074 can bind to the promoters of _BBX18_ (_AT2G21320_), _SOBIR1_ (_AT2G31880_) and _CPNB2_ (_AT3G13470_) via NRS1, NRS2 or MybSt1 in _Arabidopsis_, respectively. The function of _BBX18_ and _SOBIR1_ in _Arabidopsis_ response to stress has been confirmed. Heat stress responsive gene _BBX18_ plays a negative role in thermotolerance [42]. Under-expression of _BBX18_ displayed both increased basal and acquired thermotolerance in the transgenic plants, while over-expression of _BBX18_ reduced tolerance to heat stress in transgenic lines [42]. At the same time, the expression of _ANAC074_ was down-regulated gradually by heat (37 \({}^{\circ}\)C) treatment compared with the normal condition (22 \({}^{\circ}\)C) (see Affymetrix Arabidopsis ATH1 Genome Array, GSE4062), which suggested ANAC074, as an potential upstream regulator of _BBX18_, is likely to play an important role under heat stress. In addition, _SOBIR1_ (_AT2G31880_) participates in regulating biotic stress response including _Magnuporthe oryzae_[43] and fungal infection [44]. The function of _SOBIR1_ in biotic stress has been confirmed and whether _ANAC074_ also plays a role in biotic stress requires further studies.