BES/BZR Transcription Factor TaBZR2 Positively Regulates Drought Responses by Activation of TaGST1

Abstract

BRI1-EMS suppressor (BES)/brassinazole-resistant (BZR) family transcription factors are involved in a variety of physiological processes, but the biological functions of some BES/BZR transcription factors remain unknown; moreover, it is not clear if any of these proteins function in the regulation of plant stress responses. Here, wheat (Triticum aestivum) brassinazole-resistant 2 (TaBZR2)-overexpressing plants exhibited drought tolerant phenotypes, whereas downregulation of TaBZR2 in wheat by RNA interference resulted in elevated drought sensitivity. electrophoretic mobility shift assay and luciferase reporter analysis illustrate that TaBZR2 directly interacts with the gene promoter to activate the expression of T. aestivum glutathione s-transferase-1 (TaGST1), which functions positively in scavenging drought-induced superoxide anions (O₂ ^-). Moreover, TaBZR2 acts as a positive regulator in brassinosteroid (BR) signaling. Exogenous BR treatment enhanced TaBZR2-mediated O₂ ^- scavenging and antioxidant enzyme gene expression. Taken together, we demonstrate that a BES/BZR family transcription factor, TaBZR2, functions positively in drought responses by activating TaGST1 and mediates the crosstalk between BR and drought signaling pathways. Our results thus provide new insights into the mechanisms underlying how BES/BZR family transcription factors contribute to drought tolerance in wheat.

Figure 1.

Expression and localization of TaBZR2 in wheat under BR and drought conditions. A and B, The expression profile of TaBZR2 in 2-week–old wheat seedling leaf and root tissue under drought and BR treatments for the indicated time. Transcript levels were quantified by RT-qPCR assays. The expression of β-actin was analyzed as internal control. Each data point is the mean (±se) of three experiments. C and D, Protein level of TaBZR2 in 2-week–old wheat seedlings after drought and BR treatments for the indicated time. Total proteins were extracted and subjected to immunoblot analysis with anti-TaBZR2 antibodies. Rubisco was used as a loading control. E, Localization of TaBZR2 protein under drought and BR conditions. The nuclear/cytoplasmic signal ratio represents nuclear-accumulated TaBZR2 versus cytoplasmic-accumulated TaBZR2. Images were observed under a laser scanning confocal microscope. Scale bar = 12 μm. Each data point is the mean (±se) of 10 biological replicates (**P < 0.01; Student’s t test).

Figure 2.

TaBZR2-overexpressing wheat plants exhibit improved drought tolerance. A, Phenotypes of TaBZR2-overexpressing (OE5, OE9, and OE11) and wild-type wheat plants under well-watered and drought conditions. B, RT-qPCR analysis of TaBZR2 gene expression in TaBZR2-overexpressing and wild-type plants. The expression of β-actin was analyzed as an internal control. Each data point is the mean (±se) of three experiments. C, Survival rate of the control and water-stressed plants (without irrigation for 21 d). D, Pro content in seedlings under normal and drought conditions. E, Electrolyte leakage in seedlings under normal and drought conditions. F, MDA content in seedlings under normal and drought conditions. Each data point is the mean (±se) of three experiments (10 seedlings per experiment). The asterisks indicate significant differences between TaBZR2-overexpressing and wild-type wheat plants (Student’s t test, *P < 0.05 and **P < 0.01). WT, wild type.

Figure 3.

TaBZR2-RNAi wheat plants show enhanced drought sensitivity. A, Phenotypes of TaBZR2-RNAi (Ri3 and Ri7) and wild-type wheat plants under well-watered and drought conditions. B, RT-qPCR analysis of TaBZR2 gene expression in TaBZR2-RNAi and wild-type plants. The expression of β-actin was analyzed as an internal control. Each data point is the mean (±se) of three experiments. C, Survival rate of the control and water-stressed plants (without irrigation for 18 d). D, Pro content in seedlings under normal and drought conditions. E, Electrolyte leakage in seedlings under normal and drought conditions. F, MDA content in seedlings under normal and drought conditions. Each data point is the mean (±se) of three experiments (10 seedlings per experiment). The asterisks indicate significant differences between TaBZR2-RNAi and wild-type wheat plants (Student’s t test, *P < 0.05 and **P < 0.01). WT, wild type.

Figure 4.

Analysis of the expression levels of TaBZR2 downstream genes. A, Venn diagrams comparing the up- and downregulated genes between wild-type plants under normal and drought conditions (WTN and WTD), and TaBZR2-overexpressing and wild-type plants under normal (TaBZR2-OEN/WTN) and drought conditions (TaBZR2-OED/WTD). B, Functional categorization analysis of candidate TaBZR2 target genes in biological process under drought conditions. C, The expression levels of drought-responsive genes in TaBZR2-overexpressing, TaBZR2-RNAi, and wild-type wheat plants. Two-week–old wheat seedlings treated with 15% (w/v) PEG 6000 for 6 h were used for RNA isolation. Transcript levels were quantified by RT-qPCR assays, and the expression of β-actin was used as an internal control. Each data point is the mean (±se) of three experiments (10 seedlings per experiment). WT, wild type.

Figure 5.

TaBZR2 functions positively in scavenging drought-induced O₂⁻. A, NBT staining in primary root tips of TaBZR2-RNAi and wild-type wheat plants grown in half-strength Hoagland’s liquid medium, medium containing 15% (w/v) PEG 6000, medium containing 1 mm of DMTU, or medium containing 15% (w/v) PEG 6000 + 1 mm of DMTU for 72 h. The strength of color shows the concentration of O₂⁻ in the root tips. Scale bar = 1 mm. B, Measurements of the O₂⁻ contents of TaBZR2-RNAi and wild-type wheat plants grown in half-strength Hoagland’s liquid medium, medium containing 15% (w/v) PEG 6000, medium containing 1 mm of DMTU, or medium containing 15% (w/v) PEG 6000 + 1 mm of DMTU for 72 h. Each data point is the mean (±se) of six biological replicates. The asterisks indicate significant differences between TaBZR2-RNAi and wild-type wheat plants (Student’s t test, **P < 0.01). C, Phenotypes of TaBZR2-RNAi and wild-type wheat plants grown in half-strength Hoagland’s liquid medium, medium containing 15% PEG 6000, medium containing 1 mm of DMTU, or medium containing 15% (w/v) PEG 6000 + 1 mm of DMTU. Scale bar = 2 cm. D, Measurement of the total fresh weight of TaBZR2-RNAi and wild-type wheat plants grown in half-strength Hoagland’s liquid medium, medium containing 15% (w/v) PEG 6000, medium containing 1 mm of DMTU, or medium containing 15% (w/v) PEG 6000 + 1 mm of DMTU. Each data point is the mean (±se) of six biological replicates. The asterisks indicate significant differences between TaBZR2-RNAi and wild-type wheat plants (Student’s t test, *P < 0.05). WT, wild type.

Figure 6.

TaBZR2 directly regulates the expression of TaGST1. A, The diagram shows the structure of the TaGST1 promoter. The sequences represent TaGST1 probe sequences. The underlined sequences indicated the core elements or mutated core elements in the TaGST1 probe. B, EMSA of TaBZR2 binding to the promoter of TaGST1. Biotin-labeled TaGST1 probes (normal and mutated) were incubated with GST or GST-TaBZR2 protein. 100× competitor fragments were added to analyze the specificity of binding. C, TaBZR2 increases TaGST1 promoter activity in wheat protoplasts. TaBZR2 was cotransfected with either TaGST1 promoter or mutated TaGST1 promoter. The LUC to REN ratio is shown and indicates the activity of the transcription factors on the expression level of the promoters. Each data point is the mean (±se) of 10 biological replicates (**P < 0.01; Student’s t test).

Figure 7.

TaGST1 overexpression promotes drought tolerance in transgenic wheat. A, Phenotypes of TaGST1-overexpressing and wild-type plants under normal and drought conditions. B, Survival rate of control and water-stressed plants (15% [w/v] PEG 6000 treatment for 14 d). Each data point is the mean (±se) of three experiments (10 seedlings per experiment). C, NBT staining in primary root tip of TaGST1-overexpressing and wild-type seedlings with 0 or 15% (w/v) PEG 6000 treatment for 72 h. The strength of color shows the concentration of O₂⁻ in the root tips. Scale bar = 1 mm. D, Measurements of the O₂⁻ contents of TaGST1-overexpressing and wild-type plants under normal and drought conditions. Each data point is the mean (±se) of six biological replicates. E, RT-qPCR analysis of TaGST1 gene expression in TaGST1-overexpressing and wild-type wheat seedlings. The expression of β-actin was used as an internal control. Each data point is the mean (±se) of three experiments (10 seedlings per experiment). The asterisks indicate significant differences between TaGST1-overexpressing and wild-type plants (Student’s t test, **P < 0.01). WT, wild type.

Figure 8.

TaBZR2 is a positive regulator in the BR signaling pathway. A, Phenotypes of TaBZR2-overexpressing (OE5, OE9, and OE11), TaBZR2-RNAi (Ri3 and Ri7), and wild-type wheat plants grown in half-strength Hoagland’s liquid medium or medium containing 1 μm of BR. Scale bar = 2 cm. Root length of TaBZR2-overexpressing, TaBZR2-RNAi, and wild-type wheat plants grown on half-strength Hoagland’s medium that contained different concentrations of BR (0, 0.25, or 1 μm) in the light for 7 d. Each data point is the mean (±se) of three experiments (20 seedlings per experiment). The asterisks indicate significant differences between TaBZR2 transgenic (TaBZR2-overexpressing lines and TaBZR2-RNAi lines) and wild-type plants (Student’s t test, *P < 0.05). B, EMSA of TaBZR2 binding to the BRREs in the promoter of TaD2. Biotin-labeled BRRE probes (normal and mutated) were incubated with GST or GST-TaBZR2 protein. 100× competitor fragments were added to analyze the specificity of binding. C, The expression levels of BR biosynthetic genes in TaBZR2 transgenic (TaBZR2-overexpressing lines and TaBZR2-RNAi lines) and wild-type plants. The expression of β-actin was used as an internal control. Each data point is the mean (±se) of three experiments (10 seedlings per experiment). WT, wild type.

Figure 9.

TaBZR2 regulates wheat drought tolerance through the BR-dependent pathway. A, The expression levels of stress-responsive genes in TaBZR2 transgenic (TaBZR2-overexpressing lines and TaBZR2-RNAi lines) and wild-type plants grown in half-strength Hoagland’s liquid medium, medium containing 15% (w/v) PEG 6000, medium containing 10 nm of BR, or medium containing 15% (w/v) PEG 6000 + 10 nm of BR for 6 h. Each data point is the mean (±se) of three experiments (10 seedlings per experiment). B, Protein level of TaBZR2 in TaBZR2-overexpressing, TaBZR2-RNAi, and wild-type wheat plants upon drought and BR treatments for 6 h. Total proteins were extracted and subjected to immunoblot analysis with anti-TaBZR2 antibodies. Rubisco was used as a loading control. C, NBT staining in primary root tip of TaBZR2-overexpressing, TaBZR2-RNAi, and wild-type wheat plants grown in half-strength Hoagland’s liquid medium, medium containing 15% (w/v) PEG 6000, medium containing 10 nm of BR, or medium containing 15% (w/v) PEG 6000 + 10 nm of BR for 72 h. The strength of color shows the concentration of O₂⁻ in the root tips. Scale bar = 1 mm. D, Measurements of the O₂⁻ contents of TaBZR2-overexpressing, TaBZR2-RNAi, and wild-type wheat plants grown in half-strength Hoagland’s liquid medium, medium containing 15% (w/v) PEG 6000, medium containing 10 nm of BR, or medium containing 15% (w/v) PEG 6000 + 10 nm of BR for 72 h. Each data point is the mean (±se) of six biological replicates. The asterisks indicate significant differences between TaBZR2 transgenic (TaBZR2-overexpressing lines and TaBZR2-RNAi lines) and wild-type plants (Student’s t test, **P < 0.01). WT, wild type.