Cloning and sequence analysis of DREB
As shown in Fig. S1, the CDS sequence of DREB2, DREB6 and Wdreb2 in wheat AK58 was 732 bp, 837 bp and 1035 bp respectively, DREB2 and Wdreb2 had no intron, but one 712 bp intron was found in DREB6. CD-search analysis indicated that the typical AP2/EREBP conserved domain was found in amino acid sequence of DREB2, DREB6 or Wdreb2 (Fig. S1), was composed of YRG and RAYD conserved modules with three β folds and one α helix, simultaneously, valine (V) and glutamate (E) were very conserved at 14 th or 19 th of AP2/EREBP domain (Fig. 1, a). The nucleotide sequences or amino acid sequences of DREB2, DREB6 and Wdreb2 were further compared by DNAMAN, the similarity among amino acid sequences was low with only 33.24% identity (Fig. 1, b), but AP2/EREBP domains had 73.25% identity, even reached to 83.93% between AP2/EREBP domains of DREB6 and Wdreb2 (Fig. 1, a).
Homologous sequences of DREB2, DREB6 and Wdreb2 from wheat AK58 were analyzed and compared (Table 1, Fig. S2), the similarity of wheat DREB2 was 95% with Aegilops tauschii ERF, and was about 60% with TINY from Oryza sativa, Sorghum bicolor or Zea mays, AP2/EREBP domain of DREB2 was the same to that of Aegilops tauschii ERF and Zea mays TINY (Fig. S2, a). As listed in Table 1, the similarity of wheat DREB6 and some sequences was higher and was 98% or so, such as Thinopyrum elongatum AP2/EREBP, Aegilops biuncialis DREB2, Leymus multicaulis DREB2, etc. AP2/EREBP domain of DREB6 was the same to that of Thinopyrum elongatum AP2/EREBP, Aegilops biuncialis DREB2 and Agropyron mongolocum AP2/EREBP (Fig. S2, b). In addition, the similarity of wheat Wdreb2 with Aegilops tauschii DREB2B reached up to 99%, was also higher and was about 95% with Aegilops speltoides DREB1, Triticum turgidum DRF or Triticum dicoccoides DREB (Table 1). Furthermore, AP2/EREBP domain of Wdreb2 was the same to that of Aegilops tauschii DREB2B and Aegilops speltoides DREB1 (Fig. S2, c).
The expression pattern of DREB in wheat
As shown in Fig. 2, under normal condition, the expression level of DREB2, DREB6 and Wdreb2 in leaf was obviously higher than that in root, which was especially significant in Wdreb2 (P<0.05). Compared with that of DREB6, the expression level of Wdreb2 was significantly higher, yet the expression level of DREB2 was lower. Under drought stress, the expression level of DREB2, DREB6 and Wdreb2 in leaf was also higher than that in root (P<0.05), compared with the control, the expression of DREB2, DREB6 and Wdreb2 altered, but this change was different along with the increase of stress time.
Under drought stress, the expression level of DREB2 increased, and reached to the highest level as stressed for 2 h, which was significantly higher than the control (P<0.05), however the expression level of DREB2 decreased along with the increase of stress time, and was lower as stressed for 8-10 h, which was still higher than the control (P<0.05) (Fig. 2, a). The expression level of DREB6 was also the highest as stressed for 2 h, and was significantly higher than the control (P<0.05). Subsequently, along with the increase of stress time, the expression level of DREB6 gradually decreased, was significantly lower than the control as stressed for 10-12 h (P<0.05) (Fig. 2, b). As shown in Fig. 2 (c), the expression level of Wdreb2 in root significantly increased under drought stress, was obviously higher the control as stressed for 2 h, and also significantly increased in leaf when stressed for 6-8 h, especially stressed for 12 h (P<0.05).
Promoter analysis of wheat DREB
In this study, the promoter of DREB2, DREB6 and Wdreb2 was cloned, was respectively 1735 bp, 1792 bp or 649 bp, and was submitted to GenBank (MT974473, MT974471, MT974472). As shown in Fig. 3 and Table S1-S3, the promoter of DREB2, DREB6 and Wdreb2 contained basic regulatory element, such as TATA-box, CAAT-box, and there were 26, 18 and 5 TATA-boxes in the promoter of DREB2, DREB6 or Wdreb2, respectively. Many elements related to adverse stress were also found in the promoter of DREB2, DREB6 and Wdreb2, such as drought response element DRE/CRT, low temperature response element LTR, abscisic acid response element ABRE, light response element GAG-motif, drought-induced element MYB binding sites, etc (Fig. 3, Table S1-S3).
Further analysis found that there were some unique elements in the promoter of DREB2, DREB6 or Wdreb2, for example, the promoter of DREB2 had specially light response element MNF, leaf development element HD-ZIP and meristem specificity element OCT (Fig. 3, a; Table S1). A series of specific functional elements were also found in the promoter of DREB6, such as ethylene response element ERE, fungal elicitor response element Box-W1, MeJA regulatory element CGTCA-motif, and gibberellin response element P-box (Fig. 3, b; Table S2). Moreover, the promoter of Wdreb2 had root specificity element as1, zein metabolism regulation element O2-site, light response element C-box, and CE3 element involved in ABA and VP1 reactions (Fig. 3, c; Table S3).
Methylation analysis of DREB promoter
The distribution of CpG island in the promoter of DREB2, DREB6 and Wdreb2 was predicated and analyzed by MethPrimer and EMBOSS CpG Plot, one CpG island with 234 bp was found in the promoter of DREB2 (Fig. S3, a). As shown in Fig. S3 (b), four CpG islands located respectively in 507-644 bp, 826-960 bp, 1149-1584 bp or 1631-1735 bp of DREB6 promoter, and one CpG island with 559 bp existed in the promoter of Wdreb2 (Fig. S3, c). Furthermore, there were also functional elements in above CpG islands, such as abscisic acid response element, light response element, low temperature response element, and so on (Fig. 3, Table S1-S3).
Some CpG islands predicted in the promoter of DREB2, DREB6 and Wdreb2 were further examined from wheat leaf by bisulfite sequencing PCR (BSP), and found that there were more CHH sites and less CHG sites in the promoter region of DREB2, DREB6 and Wdreb2, but methylation rate of CG was the highest (Fig. 4, Table 2). In the promoter region of DREB2, CHH sites were not methylated, methylation rate of CG and CHH was 2.38% or 1.03%, and belonged to mild methylation (<20%) (Fig. 4, a; Table 2). As shown in Fig.4 (b) and Table 2, in the promoter region of DREB6, methylation rate of CG was 88.08% and was severely methylated (>60%), methylation rate of CHG was 51.36% and was moderately methylated (>20%), but methylation rate of CHH was only 4.93% and belonged to mild methylation (< 20%). Furthermore, in the promoter region of Wdreb2, methylation rate of CG, CHG or CHH was 1.89%, 1.0% and 0.29%, respectively, which were all mildly methylated (Fig.4, c; Table 2).
Methylation level of DREB promoter under drought stress
Under drought stress, cytosine methylation altered in the promoter region of DREB2, DREB6 and Wdreb2 from wheat leaf (Fig. 5). Compared with the control, methylation rate of CG in the promoter region of DREB2 decreased obviously (P<0.01), was 0.5% or 1.42% as stressed for 2 h and 10 h, but methylation rate of CHG and CHH increased significantly as stressed for 10 h (P<0.01). Further analysis showed that methylation level of DREB2 promoter was obviously lower or higher than the control when stressed for 2 h or 10 h, and this difference was significant (P<0.05) (Fig. 6, a).
As shown in Fig. 6 (b), methylation level of DREB6 promoter changed under drought stress, and was significantly higher than the control when stressed for 12 h (P<0.05). Compared with the control, methylation rate of CG and CHG was obviously lower or higher as stressed for 2 h and 12 h, although methylation rate of CG and CHG was significantly lower as stressed for 2 h, the promoter region of DREB6 was still heavily CG cytosine methylated (>60%) and moderately CHG cytosine methylated (>20%). As stressed for 2 h or 12 h, methylation rate of CHH was higher than the control, but this change was less than that of CG and CHG (P<0.05).
Furthermore, methylation level of Wdreb2 promoter also changed under drought stress, was significantly higher or lower than the control when stressed for 2 h and 12 h (P<0.01) (Fig. 6, c). Methylation rate of CG, CHG and CHH was respectively 2.16%, 1.5% or 1.02% as stressed for 2 h, and was obviously higher than the control (P<0.01), however was significantly lower than the control as stressed for 12 h (P<0.01).
Methylation status in DREB promoter under drought stress
As listed in Table 3, methylation status in the promoter region of DREB2, DREB6 and Wdreb2 had significant change under drought stress. Along with the increase of stress time, the number of hypermethylation sites significantly increased in DREB2 promoter, for example, there were 1 CG site and 2 CHH sites in hypermethylation status as stressed for 2 h, but were 2 CG sites, 3 CHH sites and 1 CHG site as stressed for 10 h, furthermore, there were 3 CG sites and 1 CHH site in demethylation status under drought stress.
Under drought stress, the number of hypermethylation and demethylation sites also changed in DREB6 promoter (Table 3), as stressed for 2 h, 8 CHH sites and 1 CHG site were hypermethylated, 7 CHH sites and 1 CG site were demethylated, however there were 10 CHH sites, 1 CHG site and 1CG site in hypermethylation status, 8 CHH sites, 1CG site and 1CHG site were in demethylation status as stressed for 12 h (Table 3). Along with the increase of stress time, the number of hymethylation sites had hardly changed in Wdreb2 promoter, but demethylation sites increased, and the change of methylation status was significant in CHH site, after stressed for 2 h, 2 CHH sites were respectively hypermethylated and demethylated, there were 1 CHH site in hypermethylation status and 2 CHH sites in demethylation status as stressed for 12 h (Table 3).
Correlation analysis between promoter methylation and expression of DREB
The correlation between promoter methylation and expression of DREB2, DREB6 or Wdreb2 in wheat leaf was analyzed by SPSS software. As listed in Table S4, Pearson coefficient r between expression of Wdreb2 and methylation rate of CG, CHG or CHH was respectively -0.986, -0.973 and -0.878, indicating that significant negative correlation existed between promoter methylation and gene expression of Wdreb2, similarly, promoter methylation and gene expression of DREB6 was negatively correlated (Table S4). Although significant negative correlation existed between expression of DREB2 and methylation rate of CG or CHG (Table S4), but promoter methylation of DREB2 had no negative correlation with its expression as stressed for 10 h (Fig. 2, a; Fig. 6, a).