Cloning and sequence analysis of DREBs
As shown in Fig. S1, CDS sequences of DREB2, DREB6 and Wdreb2 in wheat AK58 comprise 732 bp, 837 bp and 1035 bp respectively, DREB2 and Wdreb2 have no intron, but one 712-bp intron was found in DREB6. A typical AP2/EREBP domain was identified in all deduced protein sequences of three DREB genes using the NCBI CD-search tool (Fig. S1). AP2/EREBP domain contains YRG and RAYD conserved modules with three β folds and one α helix, valine (V) and glutamate (E) are highly conserved at 14th or 19th residue of AP2/EREBP domain (Fig. 1, a). The amino acid sequences of DREB2, DREB6 and Wdreb2 were further compared and analyzed, despite the low overall sequence similarity among three DREBs (33.24% identity) (Fig. 1, b), AP2/EREBP domains in three DREBs had 73.25% identity, even up to 83.93% between DREB6 and Wdreb2 (Fig. 1).
The deduced protein sequences of DREB2, DREB6 and Wdreb2 from wheat AK58 were also aligned with their homologous sequences, the sequence identity between wheat DREB2 and Aegilops tauschii ERF was 95%, but only around 60% identity was determined between wheat DREB2 and other homologous sequences (Table 1), and AP2/EREBP domain of wheat DREB2 was the same to that of Aegilops tauschii ERF (Fig. S2, a). As listed in Table 1, wheat DREB6 showed higher similarity to some homologous sequences (97% identity or so), its AP2/EREBP domain had higher identity with that of Aegilops biuncialis DREB2, Agropyron mongolocum AP2/EREBP, Dasypyrum villosum DREB and Leymus multicaulis DREB2 (Fig. S2, b). The higher sequence similarity (99% identity) was observed between wheat Wdreb2 and Aegilops tauschii DREB2B, wheat Wdreb2 also exhibited about 94% identity to Aegilops speltoides DREB1, Triticum turgidum DRF, Triticum dicoccoides DREB or Triticum urartu DREB2B (Table 1). Furthermore, AP2/EREBP domain of Wdreb2 was identical to that of Aegilops tauschii DREB2B and Triticum turgidum DRF 1 (Fig. S2, c).
Table 1 Homologous amino acid sequences of wheat DREBs by BLASP
|
Protein
|
Accession Number
|
Identity
|
DREB2
|
Aegilops tauschii ERF
|
XP-020183719.1
|
95%
|
Setaria italica ERF
|
XP-004968548.2
|
66%
|
Oryza sativa TINY
|
XP-015644400.1
|
64%
|
Brachypodium distachyon ERF
|
XP-010233006.1
|
64%
|
Dichanthelium oligosanthes DREB3
|
OEL19602.1
|
61%
|
Sorghum bicolor TINY
|
XP-002454993.1
|
61%
|
Zea mays TINY
|
XP-020398183.1
|
59%
|
DREB6
|
Thinopyrum elongatum AP2/EREBP
|
AEI98920.1
|
98%
|
Triticum aestivum DREBW73
|
AAY44604.1
|
98%
|
Agropyron mongolocum AP2/EREBP
|
AJD80690.1
|
94%
|
Aegilops biuncialis DREB2
|
CBX87024.1
|
97%
|
Leymus multicaulis DREB2
|
AFO12475.1
|
97%
|
Thinopyrum bessarabicum DREB
|
AIY22662.1
|
96%
|
Dasypyrum villosum DREB
|
AIY22669.1
|
97%
|
Wdreb2
|
Aegilops tauschii DREB2B
|
XP-020156298.1
|
99%
|
Triticum aestivum DREB5B
|
AAX13287.1
|
99%
|
Aegilops speltoides DREB1
|
AC035588.1
|
96%
|
Triticum turgidum DRF
|
AFO10996.1
|
95%
|
Triticum aestivum DREB4B
|
AAX13283.1
|
94%
|
Triticum dicoccoides DREB
|
ADM93284.1
|
93%
|
Triticum urartu DREB2B
|
EMS45041.1
|
93%
|
The homologous sequences of wheat DREB2, DREB6 and Wdreb2 were retrieved with BLASTP algorithm, which are mainly from some species in Gramineae. The homologous sequences having higher identity with wheat DREBs were selected from some Genera in Gramineae, and were further analyzed.
The expression patterns of DREBs in wheat
As shown in Fig. 2, the expression levels of DREB2, DREB6 and Wdreb2 in leaves were significantly higher than that in roots except for the expression of Wdreb2 in seedlings stressed for 2 h, in which more Wdreb2 transcripts were accumulated in roots (P<0.05). Under drought stress, the expression of DREB2, DREB6 and Wdreb2 altered, and had its own unique expression profile (Fig. 2).
The expression of DREB2 displayed similar trends in roots and leaves (Fig. 2, a), DREB2 transcript abundance increased to higher level after stressed for 2 h (P<0.01), then decreased, and was lower as stressed for 8-10 h, which was still higher in leaves than the control (P<0.05). A significant rise in DREB6 transcript level was also observed after stressed for 2 h (P<0.01), yet DREB6 subsequently showed the declined expression, which was significantly lower in leaves than the control as stressed for 8-12 h (Fig. 2, b). As shown in Fig. 2 (c), under drought stress, Wdreb2 was up-regulated in roots, especially after stressed for 2 h (P<0.01). Compared with the control, Wdreb2 transcript level in leaves altered significantly, and increased as stressed for 6-8 h, especially stressed for 12 h (P<0.01).
Promoter analysis of wheat DREB genes
In this study, the promoters of DREB2, DREB6 and Wdreb2 were cloned, and submitted to GenBank (MT974473: 1735 bp, MT974471: 1792 bp, MT974472: 649 bp). As shown in Fig. 3 and Table S1-S3, the promoters of wheat DREB genes contain basic regulatory elements, such as TATA-box, CAAT-box, there are 13, 10 and 5 TATA-boxes in the promoters of DREB2, DREB6 and Wdreb2, respectively. Many elements related to stresses were also found in the promoters 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 showed that there were some unique elements in the promoters of DREB2, DREB6 and Wdreb2. For example, light response element MNF, leaf development element HD-ZIP and meristem specificity element OCT are specifically present in the promoter of DREB2 (Table S1). A series of specific functional elements were also identified in the promoter of DREB6, such as ethylene response element ERE, fungal elicitor response element W-box and MeJA regulatory element CGTCA-motif (Table S2). Moreover, root specificity elements as1, zein metabolism regulation element O2-site, light response element C-box, and CE3 element involved in ABA and VP1 reactions were detected in the promoter of Wdreb2 (Table S3).
Promoter methylation analysis of DREB genes
The distribution of CpG island in the promoter regions of whet DREBs were predicated and analyzed using EMBOSS CpG Plot. One CpG island is present in the promoter of DREB2, DREB6 or Wdreb2, its length is 234 bp, 436 bp and 559 bp, respectively (Fig. S3). These putative CpG islands are preceded by some functional elements, such as abscisic acid responsive element, light responsive element, low-temperature responsive element and so on (Fig. 3, Table S1-S3).
Some CpG island regions with higher CG percent were further identified in wheat leaves using bisulfite sequencing PCR (BSP) (Fig. S3). As shown in Fig. 4 and Table 2, the majority of methylation sites were in CHH context among the three sequence contexts (CG, CHH and CHG, H = A, T, or C) in the promoter regions of DREB2, DREB6 and Wdreb2, but DNA methylation had a strong preference to CG context. In the promoter region of DREB2, methylation in CHH site was not detected, methylation rates of CG and CHH sites were 2.38% and 1.03%, belonging to mild methylation (<20%) (Fig. 4, a; Table 2). Fig. 4 (b) and Table 2 showed dense methylation (>60%) at CG site (88.08%), moderate methylation (>20%) at CHG site (51.36%) and mild methylation at CHH site (4.93%) in the promoter region of DREB6. In the promoter region of Wdreb2, methylation rates of CG, CHG and CHH sites were 1.89%, 1.0% and 0.29%, respectively, which were all mildly methylated (Fig. 4, c; Table 2).
Table 2 Methylation analysis of promoter regions in wheat DREB genes
Gene
|
Pattern
|
Pattern frequency
(%)
|
Methylation rate
(%)
|
Total Methylation rate (%)
|
DREB2
|
CG
|
19.09
|
2.38
|
1.17
|
|
CHG
|
11.82
|
0.00
|
|
CHH
|
69.09
|
1.03
|
DREB6
|
CG
|
25.93
|
88.08
|
31.89
|
|
CHG
|
11.11
|
51.36
|
|
CHH
|
62.96
|
4.93
|
Wdreb2
|
CG
|
29.60
|
1.89
|
0.88
|
|
CHG
|
16.00
|
1.00
|
|
CHH
|
54.40
|
0.29
|
Methylation level of DREB promoters under drought stress
Under drought stress, cytosine methylation altered in the promoter regions of DREB2, DREB6 and Wdreb2 from wheat leaves (Fig. 5). Compared with the control, methylation rate at CG site in the promoter region of DREB2 decreased obviously as stressed for 2 h (0.5%) and 10 h (1.42%) (P<0.01), but methylation rates at CHG and CHH sites increased significantly after stressed for 10 h (P<0.01). The overall methylation level of DREB2 promoter was significantly lower as stressed for 2 h but higher as stressed for 10 h than that in untreated leaves (Fig. 6, a).
As shown in Fig. 6 (b), after stressed for 2 h, methylation rates at CG and CHG sites in the promoter of DREB6 decreased significantly, but the promoter region of DREB6 was still heavily CG cytosine methylated (>60%) and moderately CHG cytosine methylated (>20%). The significantly increased methylation level of DREB6 promoter was observed as stressed for 12 h, which occurred in all three contexts (CG, CHG and CHH). Fig. 6 (c) displayed the changes in promoter methylation of Wdreb2 under drought stress. Compared with the control, the overall methylation level was significantly higher as stressed for 2 h (P<0.01), and methylation rates at CG, CHG and CHH sites were respectively 2.16%, 1.5% and 1.02% , however longer duration of treatment (12 h) led to the decreased methylation level (P<0.01).
Methylation status in DREB promoters under drought stress
As listed in Table 3, methylation status in the promoter regions of DREB2, DREB6 and Wdreb2 were significantly changed under drought stress. The demethylation at 3 CG sites and 1 CHH site in the promoter of DREB2 were detected, and longer duration of stress treatment resulted in enhanced hypermethylation of DREB2 promoter at CG, CHG and CHH sites
Table 3 Methylation patterns in promoter regions of wheat DREBs under drought stress
Gene
|
Type of
cytosine
|
No. of cytosine
|
No. of methylation site
|
Hypermethylation site
|
Demethylation site
|
CK-T1
|
CK-T2
|
CK-T1
|
CK-T2
|
DREB2
|
CG
|
21
|
1
|
2
|
3
|
3
|
CHG
|
13
|
0
|
1
|
0
|
0
|
CHH
|
76
|
2
|
3
|
1
|
1
|
DREB6
|
CG
|
34
|
0
|
1
|
1
|
1
|
CHG
|
15
|
1
|
1
|
0
|
1
|
CHH
|
84
|
8
|
10
|
7
|
8
|
Wdreb2
|
CG
|
37
|
0
|
1
|
1
|
2
|
CHG
|
20
|
0
|
0
|
1
|
1
|
CHH
|
68
|
2
|
1
|
2
|
2
|
CK-T1 and CK-T2 represent the change of methylation status in the promoter regions of wheat DREB genes under the stress of 15% PEG6000 solution as compared to the control (CK). T1 and T2 denote methylation status in the promoter region of DREB6 or Wdreb2 after wheat seedlings had been stressed for 2 h and 12 h, respectively, or represented methylation status in the promoter region of DREB2 as stressed for 2 h and10 h.
After stressed for 2 h, 8 CHH sites and 1 CHG site were hypermethylated in the promoter of DREB6, 7 CHH sites and 1 CG site were demethylated. 12 h later, the increase in hypermethylation of DREB6 promoter was obseved at CG and CHH sites, and the increase in demethylation was observed at CHG and CHH sites (Table 3). Compared to CG and CHG sites, methylation status of CHH site in Wdreb2 promoter was affected more strongly by drought stress (Table 3). For example, 2 CHH sites were hypermethylated and demethylated after stressed for 2 h, respectively, 1 CHH site in hypermethylation status and 2 CHH sites in demethylation status as stressed for 12 h.
Correlation analysis between promoter methylation and expression of DREB
In order to explore the correlation between promoter methylation and expression of wheat DREB2, DREB6 and Wdreb2 under drought stress, the relative expression levels of DREBs in wheat leaves and methylation rates at CG, CHG or CHH sites in their promoter regions were respectively analyzed by Pearson correlation coefficient. As listed in Table S4, Pearson coefficient r between expression level of Wdreb2 and methylation rates at CG, CHG and CHH sites was respectively -0.986, -0.973 and -0.878, indicating that the significant negative correlation existed between promoter methylation and gene expression of Wdreb2. Similarly, promoter methylation and expression level of DREB6 was negatively correlated (Table S4). Although the 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).