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. 2019 Aug 19;19(1):366.
doi: 10.1186/s12870-019-1948-y.

The specific MYB binding sites bound by TaMYB in the GAPCp2/3 promoters are involved in the drought stress response in wheat

Lin Zhang  1 , Zhiqiang Song  1 , Fangfang Li  1 , Xixi Li  1 , Haikun Ji  1 , Shushen Yang  2
Affiliations

The specific MYB binding sites bound by TaMYB in the GAPCp2/3 promoters are involved in the drought stress response in wheat

Lin Zhang et al. BMC Plant Biol. .

Retraction in

Abstract

Background: Drought stress is one of the major abiotic stresses that affects plant growth and productivity. The GAPCp genes play important roles in drought stress tolerance in multiple species. The aim of this experiment was to identify the core cis-regulatory elements that may respond to drought stress in the GAPCp2 and GAPCp3 promoter sequences.

Results: In this study, the promoters of GAPCp2 and GAPCp3 were cloned. The promoter activities were significantly improved under abiotic stress via regulation of Rluc reporter gene expression, while promoter sequence analysis indicated that these fragments were not almost identical. In transgenic Arabidopsis with the expression of the GUS reporter gene under the control of one of these promoters, the activities of GUS were strong in almost all tissues except the seeds, and the activities were induced after abiotic stress. The yeast one-hybrid system and EMSA demonstrated that TaMYB bound TaGAPCp2P/3P. By analyzing different 5' deletion mutants of these promoters, it was determined that TaGAPCp2P (- 1312~ - 528) and TaGAPCp3P (- 2049~ - 610), including the MYB binding site, contained enhancer elements that increased gene expression levels under drought stress. We used an effector and a reporter to co-transform tobacco and found that TaMYB interacted with the specific MYB binding sites of TaGAPCp2P (- 1197~ - 635) and TaGAPCp3P (- 1456~ - 1144 and - 718~ - 610) in plant cells. Then, the Y1H system and EMSA assay demonstrated that these MYB binding sites in TaGAPCp2P (- 1135 and - 985) and TaGAPCp3P (- 1414 and - 665) were the target cis-elements of TaMYB. The deletion of the specific MYB binding sites in the promoter fragments significantly restrained the drought response, and these results confirmed that these MYB binding sites (AACTAAA/C) play vital roles in improving the transcription levels under drought stress. The results of qRT-PCR in wheat protoplasts transiently overexpressing TaMYB indicated that the expression of TaGAPCp2/3 induced by abiotic stress was upregulated by TaMYB.

Conclusion: The MYB binding sites (AACTAAA/C) in TaGAPCp2P/3P were identified as the key cis-elements for responding to drought stress and were bound by the transcription factor TaMYB.

Keywords: Drought tolerance; TaGAPCp promoter; TaMYB; Triticum aestivum.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Location of putative cis-elements in TaGAPCp2 and TaGAPCp3 promoters. Transcriptional start site (+ 1) is shown in black arrowhead
Fig. 2
Fig. 2
Analysis of TaGAPCp2 and TaGAPCp3 promoters activity under abiotic stresses. a GUS staining of TaGAPCp2 and TaGAPCp3 promoters in transiently transformed tobacco leaves. P: positive control (CaMV 35S promoter); WT: wild type. b Analysis of RLUC activity for TaGAPCp2 and TaGAPCp3 promoters in transiently transformed tobacco leaves in response to stress. c Analysis of RLUC activity for TaGAPCp3 promoter in transiently transformed tobacco leaves in response to stress. The indicated values are the average of three independent experiments. The standard deviation (SD) is indicated at each point. Significant differences between treated and untreated (control) conditions were assessed with one-sided paired t-tests (*, P < 0.05; **, P < 0.01)
Fig. 3
Fig. 3
Histochemical staining of transgenic Arabidopsis TaGAPCp2P and TaGAPCp3P. a 15-day old plant; b Heading stage plant (5–6 weeks); c silique
Fig. 4
Fig. 4
GUS enzymatic activity quantification of transgenic Arabidopsis 24-h after abiotic stresses. a Expression of the GUS gene driven by TaGAPCp2 promoter. b Expression of the GUS gene driven by TaGAPCp3 promoter. The indicated values are the average of three independent experiments. The standard deviation (SD) is indicated at each point. Significant differences between treated and untreated (control) conditions were assessed with one-sided paired t-tests (*, P < 0.05; **, P < 0.01)
Fig. 5
Fig. 5
TaGAPCp2 and TaGAPCp3 responding to drought stress treatments in Arabidopsis. a Tolerance responses of the TaGAPCp2–overexpressing (OE-2) and TaGAPCp3–overexpressing (OE-3) lines to drought stress. Drought 25d: with holding water for 25d; R7d: resumption of water for 7 d after withholding water for 25d. b Survival rates of TaGAPCp2–overexpressing (OE-2) and TaGAPCp3–overexpressing (OE-3) transgenic lines, and WT plants on day 7 after resuming water following the withholding of water for 25d. At least 100 plants were counted and averaged for each line. c RWC of WT and transgenic lines after withholding water for 15d. d The chlorophyll contents of WT and transgenic lines after withholding water for 15 d. e The MDA content of WT and transgenic lines after withholding water for 15d. Error bars indicate ±SD (n = 3, from three technical replicates). Significant differences were assessed with one-sided paired t-tests (*, P < 0.05; **, P < 0.01). Three biological experiments were performed, which produced similar results
Fig. 6
Fig. 6
TaMYB is transcriptional activator of TaGAPCp2P and TaGAPCp3P a Schematic diagram of the probes used for Electrophoretic mobility shift assays (EMSA) and fragments used for Yeast one-hybrid. b Yeast one-hybrid activity in TaGAPCp 2/3 promoter and TaMyb. c TaMYB bound MYB binding sites by EMSA.
Fig. 7
Fig. 7
Screening of the TaGAPCp2P and TaGAPCp3P. a, b Schematic representations of the constructs containing a series of deletions of the TaGAPCp2P and TaGAPCp3P. c, d The relative expressions of Renilla luciferase in transiently transformed tobacco leaves. F1 to F7 represents tobacco leaves transformed with the dual luciferase reporter vectors driven by a series of deletions of the TaGAPCp2P (C) and TaGAPCp3P (D). The indicated values are the average of three independent experiments. The standard deviation (SD) is indicated at each point. Significant differences between treated and untreated (control) conditions were assessed with one-sided paired t-tests (*, P < 0.05; **, P < 0.01)
Fig. 8
Fig. 8
Identification of fragments bound by TaMYB in these promoters. a Schematic diagrams of the effector and reporter used for transient transactivation assays in tobacco. b, c Transactivation activity reflected by RLUC activity of RLUC/FLUC ratio, B for TaGAPCp2P; C for TaGAPCp3P. Asterisks indicate the significant difference between treatment and control (*P < 0.05,**P < 0.01 or ***P < 0.001)
Fig. 9
Fig. 9
Identification of MYB-binding site bound by TaMYB in TaGAPCp2P and TaGAPCp3P. a Schematic diagram of the probes used for EMSA and fragments used for Yeast one-hybrid. b Yeast one-hybrid confirming the MBS activities. c, d TaMYB bound MBS by EMSA, B for TaGAPCp2P; C for TaGAPCp3P
Fig. 10
Fig. 10
Verification of MBS bound by TaMYB in promoter. a, b Schematic representations of the vectors designed for MBS verification. c, d The relative expression of RLUC in transiently transformed tobacco leaves with the vectors indicated in a, b. Asterisks indicate the significant difference between treatment and control (*P < 0.05,**P < 0.01 or ***P < 0.001)
Fig. 11
Fig. 11
TaMYB gene regulate the expression of TaGAPCp2 and TaGAPCp3 in wheat protoplast. a Expression patterns of TaMYB under abiotic stresse challenge of PEG, ABA and H2O2. b The transient overexpression of TaMYB promoted the ABA-induced expression of TaGAPCp2 and TaGAPCp3 in wheat protoplasts. The protoplasts were treated with 100 uM ABA, and the relative expression levels of TaGAPCp2 and TaGAPCp3 were analysed by RT-PCR. The indicated values are the average of three independent experiments. The standard deviation (SD) is indicated at each point. Significant differences between treated and untreated (control) conditions were assessed with one-sided paired t-tests (*, P < 0.05; **, P < 0.01)
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