Cloning and sequence analysis of DREB
As shown in Fig. S1, the CDS sequence of DREB2, DREB6 and Wdreb2 in wheat was 732bp, 837bp and 1035bp respectively, DREB2 and Wdreb2 had no intron, but one 712bp 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, V (valine) and E (glutamate) were very conserved at 14th or 19th of AP2/EREBP domain (Fig. 1, a). The nucleotide sequences or amino acid sequences of DREB2, DREB6 and Wdreb2 were further compared by DNAMAN, it was found that the similarity was low with only 33.24% identity among their amino acid sequences (Fig. 1, b), their AP2/EREBP domains had 73.25% identity, even the identity reached to 83.93% between AP2/EREBP domains of DREB6 and Wdreb2 (Fig. 1, a).
Homologous sequences of DREB2, DREB6 and Wdreb2 were analyzed and compared (Table 1, Fig. S2), the similarity of wheat DREB2 was 95% with Aegilops tauschii ERF, was about 60% with TINY of Oryza sativa, Sorghum bicolor or Zea mays, and AP2/EREBP domain of DREB2 was the same to that from Aegilops tauschii and Zea mays (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, Triticum aestivum DREBW73, Aegilops biuncialis DREB2 or Leymus multicaulis DREB2, and AP2/EREBP domain of DREB6 was the same to that from Thinopyrum elongatum, Aegilops biuncialis and Agropyron mongolocum (Fig. S2, b). In addition, the similarity of wheat Wdreb2 with Aegilops tauschii DREB2B reached up to 99%, was also higher with Aegilops speltoides DREB, Triticum turgidum DRF or Triticum dicoccoides DREB, and was about 95% (Table 1). Furthermore, AP2/EREBP domain of Wdreb2 was the same to that from Aegilops tauschii and Aegilops speltoides (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.
Compared with the control, the expression level of DREB2 increased under drought stress, and reached to the highest level as stressed for 2h, which was significantly higher than the control (P<0.05). Along with the increase of stress time, the expression level of DREB2 decreased, was the lowest as stressed for 10h, 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 2h, 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 10-12h, and was the lowest under drought stress for 12h (P<0.05) (Fig. 2, b). As shown in Fig. 3 (b), compared with the control, the expression level of Wdreb2 in root significantly increased under drought stress, was obviously higher the control as stressed for 2h, and the expression level of Wdreb2 in leaf also significantly increased when stressed for 6-8h, especially stressed for 12h (P<0.05).
Promoter analysis of wheat DREB
In this study, the promoter of DREB2, DREB6 and Wdreb2 was cloned, their length was respectively 1735bp, 1792bp or 649bp, and was analyzed by Plant CARE and PLACE. As shown in Fig. 3 and Table S1-S3, the promoter of DREB2, DREB6 and Wdreb2 contained basic regulatory element, such as TATA-box and CAAT-box, and there were 26, 18 and 5 TATA-boxes in the promoter of DREB2, DREB6 and 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 as, 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).
Distribution of CpG island in 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 507bp-644bp, 826bp-960bp, 1149bp-1584bp or 1631bp-1735bp of DREB6 promoter, and one CpG island with 559bp existed in the promoter of Wdreb2 (Fig. S3, c). Furthermore, there were also functional elements in the CpG island, such as abscisic acid response elements, light response elements, low temperature response element, and so on (Fig. 3, Table S1-S3).
CpG island in the promoter of DREB2, DREB6 and Wdreb2 was further examined from leaf by bisulfite sequencing PCR (BSP), and found that there were more CHH sites and less CHG sites in the promoter of DREB2, DREB6 and Wdreb2, but methylation rate of CG was the highest (Fig. S4). In the promoter 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. S4, a). As shown in Fig. S3 (b), in the promoter 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 of Wdreb2, methylation rate of CG, CHG or CHH was 1.89%, 1.0% and 0.29%, respectively, which were all mildly methylated (Fig. S4, c).
Methylation level of DREB promoter under drought stress
Under drought stress, cytosine methylation altered in the promoter of DREB2, DREB6 and Wdreb2 from leaf (Fig. 4). Compared with the control, methylation rate of CG in the promoter of DREB2 decreased obviously (P<0.01), was 0.5% or 1.42% as stressed for 2h and 10h, but methylation rate of CHG and CHH increased significantly as stressed for 10h (P<0.01), and drought stress for 2h had no effect on methylation of CHG and CHH. Further analysis showed that methylation level of DREB2 promoter was obviously lower or higher than the control when stressed for 2h or 10h, this difference was very significant (P<0.01) (Fig. 5, a).
As shown in Fig. 5 (b), methylation level of DREB6 promoter changed under drought stress, compared with the control, methylation rate of CG and CHG was obviously lower or higher as stressed for 2h and 12h (P<0.01), although methylation rate of CG and CHG was significantly lower as stressed for 2h, the promoter of DREB6 was still heavily CG cytosine methylated (>60 %) and moderately CHG cytosine methylated (>20 %). As stressed for 2h or 12h, methylation rate of CHH was obviously higher or lower than the control, but this change was less than that of CG and CHG (P<0.01), methylation level of DREB6 promoter was significantly higher when stressed for 12h (Fig. 5, b).
Furthermore, methylation level of Wdreb2 promoter also changed under drought stress, was significantly higher or lower than the control when stressed for 2h and 12h (P<0.01) (Fig. 5, c). Methylation rate of CG, CHG and CHH was respectively 2.16%, 1.5% or 1.02% as stressed for 2h, and was obviously higher than the control (P<0.01), however was significantly lower than the control as stressed for 12h (P<0.01).
Methylation status in DREB promoter under drought stress
As listed in Table 2, methylation status in the promoter 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 2h, but 2 CG site, 3 CHH sites and 1 CHG sites were in hypermethylation status as stressed for 10h, furthermore, there were 3 CG sites and 1 CHH site in demethylation status under drought stress.
Under drought stress, the number of hymethylation and demethylation sites also changed in DREB6 promoter, as stressed for 2h, 8 CHH sites and 1 CHG site were hypermethylated, 7 CHH sites and 1 CG site were in demethylation status, 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 12h (Table 2). 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 2h, 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 12h.
Correlation analysis between promoter methylation and expression of DREB
The correlation between promoter methylation and expression of DREB2, DREB6 and Wdreb2 in leaf was analyzed by SPSS software. As shown 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 12h (Fig. 2, a; Fig. 5, a).