## 2 Results and Discussion
### Binding of Two Novel Motifs and MybSt1 Motif to ANAC074  
The bindings of ANAC074 to NRS1 (TAGTT), NRS2 (GAATC), MybSt1 (GGATA) and their mutants were investigated by Y1H to investigate the specification of binding. The results showed that the ANAC074 can bind to MybSt1 element, NRS1 and NRS2, while the MybSt1 mutants (MYB-M1: TTATA; MYB-M2: GTCGA; MYB-M3: TTCGG), NRS1 mutants (NRS1-M1: GGCTT, NRS1-M2: TAGGG; NRS1-M3: GGGGG), and NRS2 mutants (NRS2-M1: TCATC; NRS2-M2: GACGC; NRS2-M3: TCCGA) all lost binding activity with ANAC074, indicating that the bindings of ANAC074 to MybSt1 \begin{table}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{**Clone Number**} & **Random DNA Insertion Sequence (5’-3’)** & \multirow{2}{*}{**Motif Prediction**} \\  & **Underlined Together with the Two Sides of** & \\  & **Flanking Sequences** & \\ \hline
1 & CCCCTTCACGCGCG & \\  & & NACS: “CACG” \\ \hline
2 & CCCAGGATAACGGG &
\begin{tabular}{c} MybSt1-binding \\ element: “GGATA” \\ \end{tabular} \\ \hline
3 & CCCTIAGTTTGGGG & LTEE-1: “CCGAAA” \\ \hline
4 & CCCTGAATCTCGGG & ARR1 AT: “NGATT” \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Analysis of the insertion sequence that binds to ANAC074. element, NRS1 and NRS2 are specific (Figure 2A). Furthermore, transient coexpression of ANAC074 and MybSt1 element, NRS1, and NRS2 or their complete mutants (NRS1-M3: GGGGG; NRS2-M3: TCCGA; MYB-M3: TICGC) in tobacco also confirmed that the ANAC074 bound to MybSt1, NRS1 and NRS2 elements specifically (Figure 2B,C). Although various cis-elements for NAC proteins have been identified [6; 13; 19; 20; 21; 22; 25], the NRS1, NRS2, and MybSt1 binding sequences for ANAC074 are different from those reported previously. These results suggested different NAC transcription factors may bind to different core element sequences. The transcription factors regulate gene expression by binding to certain motifs. Therefore, their function could be predicted by their binding motifs. ANAC074 protein can bind to other cis-acting elements, in addition to sequences containing \"CACG\" or its complement, suggesting  
Figure 1: Serial deletion of the inserted sequence to determine the motif recognized by ANAC074. **(A)** Serial deletion of the inserted sequence to determine the exact sequence of NRS1 recognized by ANAC074. **(B)** Serial deletion of the inserted sequence to determine the exact sequence of NRS2 recognized by ANAC074. pGADT7-ANAC074: pGADT7-Rec2 vector that encodes ANAC074 fused with GAL4 AD as the effector construct. pHIS2-NRS1-D0-8/NRS2-D0-8: Three tandem copies of sequence of D0 to D8 of NRS1/NRS2 were respectively cloned into the pHIS2 to drive the _HIS3_ reporter gene as the reporter constructs. The right panel shows the sequence of different NRS1 or NRS2 deletions. Positive control: pGADT7-p53 interacting with p53 HIS2. Negative control: pGADT7-ANAC074 interacting with p53 HIS2. Positive transformants were further confirmed by spotting yeast cells with serial dilutions (1/1, 1/10, 1/100, 1/1000) onto medium of SD/-His/-Leu/-Trp with 40 mM 3-AT. The transformants grown at SD/-Leu/-Trp were used as positive controls for transformant growth. that the regulatory function of ANAC074 is different from that of other NAC domain proteins like ANAC019, ANAC055, and ANAC072. Figure 2: The bindings of NRS1, NRS2 and MybSt1 to ANAC074. (**A**) Analysis of the specific bindings of NRS1, NRS2, and MybSt1 to ANAC074 using Y1H assay. The right panel showed the sequences of NRS1, NRS2, MybSt1 motifs and their respective mutants (M1, M2 and M3). Positive transformants were confirmed by spotting yeast cells with serial dilutions (1/1, 1/10, 1/100, 1/1000) onto medium of SD/-His/-Leu/-Trp with 40 mM 3-AT. Positive control: pGADT7-p53 interacting with p53 HIS2; Negative control: pGADT7-ANAC074 interacting with p53 HIS2. The transformants grown at SD/-Leu/-Trp were used as positive controls for transformant growth. (**B**) Schematic diagram of the reporter and effector vectors.