Exploring the Role of TaERF4a in Enhancing Drought Tolerance and Regulating Dehydrin WZY1-2 Gene Expression in Wheat

Abstract

Dehydrins (DHNs) belong to the second family of late embryogenesis abundant (LEA) proteins, which are widely distributed in plants. We cloned a SK₃-type DHN gene named WZY1-2 in Zheng yin 1 cultivar of Triticum aestivum. An ERF-type transcription factor TaERF4a was found to be involved in the regulation of the dehydrin WZY1-2 gene in our last report. The stress-responsive ability and dual-luciferase assay demonstrated that TaERF4a positively regulates WZY1-2 gene transcription under stress conditions. In this study, we further characterized the role of the transcription factor TaERF4a in plant drought tolerance. Arabidopsis thaliana heterologously overexpressing TaERF4a exhibited higher survival rate, increased superoxide dismutase (SOD) activity, elevated proline and chlorophyll content, and reduced malondialdehyde (MDA) content under drought conditions. Conversely, silencing TaERF4a in Chinese spring wheat using the virus-induced gene silencing (VIGS) method increased the sensitivity of plants to drought stress. Furthermore, we identified the specific binding site of TaERF4a in the WZY1-2 promoter. Electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assay demonstrated that TaERF4a activates the expression of the WZY1-2 dehydrin gene through binding to the DRE cis-element in its promoter. Taken together, the results of our study indicate that TaERF4a positively regulates the expression of the dehydrin WZY1-2 gene and enhances drought tolerance in plants.

Keywords: TaERF4a; dehydrin; dual-luciferase assay; transactivation; virus-induced gene silencing.

Figure 1

Performance of wild-type (WT) and TaERF4a-overexpressing Arabidopsis under drought and rehydration treatments; (A) phenotypic analysis of transgenic Arabidopsis plants under normal growth, drought stress conditions and after rewatering; chlorophyll content (B), SOD content (C), malondialdehyde content (D), and proline content (E) of wheat leaves from WT and transgenic plants under normal growth or drought stress conditions; and (F) leaf water loss rate of transgenic and WT plants after 10 h of drought treatment; data are presented as means ± SD of three replicates (* p < 0.05, ** p < 0.01).

Figure 2

Growth status of wild-type (WT) and TaERF4a-overexpressing Arabidopsis seedlings under PEG-induced stress; (A) the seedlings of WT and transgenic plants were grown on 1/2 MS agar medium containing 10% PEG6000; and (B) the root length were measured at about 1-week-old seedling stage. Data are presented as means ± SD of three replicates (** p < 0.01).

Figure 3

Performance of VIGS-mediated TaERF4a-silencing wheat under drought treatment; the phenotypes (A), TaERF4a and WZY1-2 gene expression levels (B), relative water content (C), chlorophyll content (D), and malondialdehyde content (E) of wheat leaves treated with BSMV:00 (empty vector), BSMV:PDS, and BSMV:TaERF4a before and after drought treatment. Data are presented as means ± SD of three replicates (** p < 0.01).

Figure 4

Transactivation activity assay of TaERF4a in yeast cells and its interaction with the promoter of the WZY1-2 gene in vitro; (A) diagram of three fragments encoding the full-length, N-terminus (1–80 amino acids), and C-terminus (81–195 amino acids) of TaERF4a, which were, respectively, introduced into the pGBKT7 vector. Transactivation assays of fusion proteins were performed in the Y2H Gold yeast strain; (B) electrophoretic mobility shift assay of transcription factor TaERF4a binding with the P1 (−718 to −462 bp), P2 (−472 to −218 bp), and P3 (−220 to +15 bp) fragments of the WZY1-2 promoter. TaERF4a-GST represents the purified fusion protein. GST was used as the negative control; and (C) EMSA analysis of TaERF4a binding to the DRE cis-acting element in the promoter segment of the WZY1-2 gene. The cis-acting elements involved include the dehydration-responsive element (DRE: GCCGAC), myb-binding site I (MBSI: TAACTG), and mutant DRE (mDRE: GTCGCA).

Figure 5

Transient luciferase activity assay of the DRE motif bound by TaERF4a in N. benthamiana leaves. Schematic diagrams of effector and reporter constructs used in the assay. The described constructs were transformed into Agrobacterium tumefaciens strain GV3101 and then infiltrated into N. benthamiana leaves. The experiments were performed with at least three biological repeats. Data are presented as means ± SD of three replicates (** p < 0.01).

Figure 6

The expression levels of stress-responsive genes CSD1 (A), POD1 (B), P5CS1 (C), RD29A (D), RAB18 (E) and DREB2A (F) in WT and transgenic Arabidopsis plants under normal and drought treatments. Data are presented as means ± SD of three replicates (** p < 0.01).

Figure 7

A working model of TaERF4a and WZY1-2 in abiotic stress response.