## 2 Results and Discussion
### Binding of NRS1, NRS2, and MybSt1 to NAC (NAM, ATAF1/2, and CUC2) Family Members  
Researchers identified 117 NAC genes in _Arabidopsis_[3]. On the basis of the phylogenetic analysis, the _Arabidopsis_ NAC family was divided into ten major polytomies by collapsing branches with bootstraps <40% [45]. To study the binding of NRS1, NRS2, and MybSt1 to other NAC members, the NACs were selected from group III (AT2G33480/ANAC041, AT1G69490/ANAC029 and AT1G77450/ANAC032), group V (AT2G43000/ANAC042), group VI (AT3G44350/ANAC061 and AT5G50820/ANAC097), group VII (AT3G04420/ANAC048 and AT4G01540/ANAC068/NTM1), group IX (AT4G28500/ANAC073/SND2), and group X (AT3G56520). Their bindings to NRS1, NRS2, and MybSt1 were studied using Y1H. The results showed that all these NACs can bind to NRS1, NRS2, and MybSt1 (Figure 6), which suggested that individual proteins belonging to different NAC groups having binding affinity for the same cis-element. This is consistent with the results of other scholars. As an example, [PERSON] et al. found that nine distantly related NAC TFs were able to bind conserved DNA target sequence with a CGT[GA] core, although with different affinities [45]. Some NACs such as AT3G56520 and AT1G77450 have less binding activity with MybSt1 motif than other NAC members, which may be related to the structure of their conserved NAC domain located at the N-terminal region. The fact that not all genes bind to the same element with the same binding capacity is reasonable, because these genes may be involved in different regulatory networks by preferring to identify different motifs.  
All in all, the knowledge of NACs that can bind to MybSt1, NRS1, and NRS2 besides NACRS will contribute to revealing the function of NACs in depth.  
Figure 5: Comparison of the binding affinities of NACRS, NRS1, NRS2, and MybSt1 to ANAC074. (**A**) Schematic diagram of the reporter and effector constructs. (**B**) Three tandem copies of NRS1, NRS2, MybSt1, or one copy of NACRS were respectively fused with minimal promoter to drive _GUS_, and co-transformed with ANAC074 under control of 35S promoter into tobacco leaves. GUS activity was determined to study their binding affinity. The empty pCAMBIA1301 was used as positive control. Co-transformed empty pROKII with pCAM-NRS1, pCAM-NRS2, pCAM-MYB, or pCAM-NACRS 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 NACRS (Student’s test; * \(p\) < 0.05).  
Figure 6: Bindings of NRS1, NRS2, and MybSt1 to other NACs from _Arabidopsis_. The bindings of ten NACs from different NAC subfamily to NRS1 (**A**), NRS2 (**B**), and MybSt1 (**C**). The ORFs of ten NACs were respectively fused with GAL4 AD in PGADT7-Rec2 as the effector constructs. pHIS2-NRS1-D4, -D7, -D6, -D8 or pHIS2-NRS2-D4, -D5, -D6, -D8: Three tandem copies of NRS1-D4, -D7, -D6, -D8, or NRS2-D4, -D5, -D6, -D8 (see Figure 1 for the sequence of these elements) were respectively cloned into the pHIS2 as the reporter constructs. Positive transformants were 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. Positive control: pGADT7-p53 interacting with p53 HIS2; Negative control: pGADT7-ANAC074 interacting with p53 HIS2.