## 2 Results and Discussion  
### Identification of the DNA Motifs Bound by ANAC074  
The TF-Centered Y1H system is a simple and efficient method for identification of the DNA sequence bound by transcription factors of interest [23; 24]. To identify the DNA sequences recognized by ANAC074, the random DNA insertion prey library was screened with ANAC074 (AT4G28530) cloned into pGADT7-Rec2 as bait using the Y1H system [23; 24]. Eight positive clones were obtained. After further selection, four positive clones were identified to have high binding affinity to ANAC074, and were sequenced. Since the two flanking sequences of the insertion sequence may also be part of cis-acting elements recognized by ANAC074, three bases of each flanking sequence together with the insertion sequence were used for further analysis. The insertion sequences were firstly predicted with PLACE and PlantCare to find the putative motifs bound by ANAC074. The insertion sequences and the predicted motifs are shown as Table 1. The sequence \"CCCCTTCACGCG\" (insertion is underlined) contains the core sequence \"CACG\". Previous studies have shown that NAC transcription factors mainly bind to the classic NACRS with discontinuous core sequence of CATGTG and CACG in promoters to regulate the expression of target genes [13; 19; 20]. In addition to discontinuous motif, some NACs can also bind continuous core sequence. _Arabidopsis_ ANAC019 and ANAC092 could bind to continuous TTNCGT[G/A] sequence [21], the conserved sequence \"CGT[G/A]\" is similar to the core sequence \"CACG\" (its reverse complementary sequence CGTG) of NACRS. _Arabidopsis_ ANAC069 protein was found to bind to C[A/G]CG[T/G] sequence, in which the sequence C[A/G]CGis similar to the core sequence \"CACG\" [22]. _Arabidopsis_ NAC1 binds to a 21-bp DNA fragment containing an as-1 element (TGACG), in which the conserved sequence \"GACG\" is also similar to the \"CACG\" [6]. In this examination, using ANAC074 as bait, one of the cis-acting elements we caught was \"CCCCTTCACGCGGG\" containing the core sequence \"CACG\" like other NAC binding elements. This result further suggested that some NAC proteins can bind core sequences \"CACG\".  
However, other NAC proteins appear to bind different sequences. [PERSON] et al. (2007) found that the _Arabidopsis_ transcriptional repressor CBNAC can bind to CBNACBS with a \"GCTT\" sequence but not the \"CACG\" sequence [25]. Therefore, we further studied the other three insertion sequences without \"CACG\". According to PLACE and PlantCare prediction, the sequence \"CCCGGATAACGGG\" includes MybSt1-binding element \"GGATA\" [26], \"CCCTGAAATCCGGG\" includes ARR1 AT motif \"NGATT\", and \"CCTTAGTTCCGG\" contains LTEE-1 motif \"CCGAAA\" (Table 1). These results implied that the ANAC074 may bind to MybSt1-binding element (shown as MybSt1 in the following), ARR1 AT and LTEE-1 motifs. To verify this hypothesis, three tandem copies of \"GGATA\", \"TGATT\", and \"CCGAAA\" were respectively cloned into pHIS2. The bindings of ANAC074 to these three sequences were investigated by Y1H assay. The results showed that the ANAC074 can bind to \"GGATA\", but cannot bind to the sequence of \"TGATT\" and \"CCGAAA\", indicating that ANAC074 binds to MybSt1 element, but cannot interact with ARR1 AT and LTEE-1 motif. Therefore, the sequence of \"CCCTTAGTTTTGG\" and \"CCCTGAAATCCCGG\" both contain unknown DNA motifs recognized by ANAC074, and were further studied.