A gain-of-function allele of a DREB transcription factor gene ameliorates drought tolerance in wheat

Abstract

Drought is a major environmental factor limiting wheat production worldwide. However, the genetic components underlying wheat drought tolerance are largely unknown. Here, we identify a DREB transcription factor gene (TaDTG6-B) by genome-wide association study that is tightly associated with drought tolerance in wheat. Candidate gene association analysis revealed that a 26-bp deletion in the TaDTG6-B coding region induces a gain-of-function for TaDTG6-BDel574, which exhibits stronger transcriptional activation, protein interactions, and binding activity to dehydration-responsive elements (DRE)/CRT cis-elements than the TaDTG6-BIn574 encoded by the allele lacking the deletion, thus conferring greater drought tolerance in wheat seedlings harboring this variant. Knockdown of TaDTG6-BDel574 transcripts attenuated drought tolerance in transgenic wheat, whereas its overexpression resulted in enhanced drought tolerance without accompanying phenotypic abnormalities. Furthermore, the introgression of the TaDTG6-BDel574 elite allele into drought-sensitive cultivars improved their drought tolerance, thus providing a valuable genetic resource for wheat breeding. We also identified 268 putative target genes that are directly bound and transcriptionally regulated by TaDTG6-BDel574. Further analysis showed that TaDTG6-BDel574 positively regulates TaPIF1 transcription to enhance wheat drought tolerance. These results describe the genetic basis and accompanying mechanism driving phenotypic variation in wheat drought tolerance, and provide a novel genetic resource for crop breeding programs.

Figure 1

Circos diagram showing the global physical distribution of the 863,030 SNPs, average nucleotide diversity, and LD blocks for each chromosome. A, Physical size of each chromosome. Wheat chromosomes are represented in different colors. B, SNP distribution is represented by SNP numbers in 1-Mb windows for each chromosome. C, Heatmap representation of the number of matched high-confidence genes in 1-Mb windows for each chromosome. D, Average nucleotide diversity, as estimated by polymorphism contents using a 1-Mb sliding window along the chromosomes. E, LD block sizes for SNPs within a 5-Mb sliding window.

Figure 2

TaDTG6-B confers natural variation in drought tolerance of wheat seedlings. A, Results of GWAS for wheat drought tolerance. The gray horizontal line indicates the genome-wide significance threshold (P = 1.38 × 10⁻⁵). B, Quantile-quantile plot for GWAS results under a general linear model (GLM) and MLM. C, Genome-wide association signals for wheat drought tolerance, shown over the 591–605 Mb region of chromosome 2B. D, Filtered gene models within the candidate region on chromosome 2B. E, SR for the two genotypes at the leading SNP on chromosome 2B. Statistical significance was determined by analysis of variance (ANOVA) (n = 302 for genotype A; n = 128 for genotype G). F, Relative expression levels of TaDTG6-B under drought stress. Values are means ± sd (n = 3).

Figure 3

The TaDTG6-B^Del574 allele is associated with drought tolerance in wheat seedlings. A, Association analysis between genetic variation at TaDTG6-B and drought tolerance. A schematic diagram of the ∼3.2-kb TaDTG6-B genomic region is shown at the top. The region encoding the AP2 domain is indicated in red. B, Pattern of pairwise LD for each polymorphism over the TaDTG6-B locus. C, Distribution of the SR for the two genotype groups based on InDel574 (Del574 and In574). D, Relative expression levels of TaDTG6-B in the two genotype groups based on InDel574. Statistical significance was determined by ANOVA. E, Schematic diagram of the primers used for two-round PCR genotyping of InDel574. F, Molecular marker (InDel574) selection of segregating NILs homozygous for either the TaDTG6-B^Del574 or TaDTG6-B^In574 alleles. G, Drought tolerance of the NILs carrying the tolerant allele TaDTG6-B^Del574 or the sensitive allele TaDTG6-B^In574. Values are means ± sd from three independent experiments; statistical significance was determined by two-sided Student’s t test (**P < 0.01).

Figure 4

Molecular characterization of the TaDTG6-B^Del574 and TaDTG6-B^In574 variants. A, Amino acid sequence alignment of TaDTG6-A, TaDTG6-B^Del574, TaDTG6-B^In574, and TaDTG6-D. The core AP2 domain is indicated with a black line and the 26-bp InDel inducing a frameshift is highlighted in the red box. B, Transient expression assays of the TaDTG6-A, TaDTG6-B^Del574, TaDTG6-B^In574, and TaDTG6-D promoters driving the transcription of the firefly LUC reporter gene in N. benthamiana leaves. C, Subcellular localization of GFP-tagged TaDTG6-A, TaDTG6-B^Del574, TaDTG6-B^In574, and TaDTG6-D in wheat protoplasts transfected with the respective encoding constructs. Scale bars, 10 µm. D, Evaluation of transactivation activity of TaDTG6-A, TaDTG6-B^Del574, TaDTG6-B^In574, and TaDTG6-D fused to the DNA-binding domain of yeast GAL4 via one-hybrid transactivation assays. The predicted conserved AP2 domain is shown in red in the schematic diagrams of the constructs (left). Yeast growth is shown (right) on SD medium lacking Trp (SD –T), Trp and His (SD –T–H), Trp, His, and Ade (SD –T, –H, –A), and SD –T, –H, –A +X-α-gal. E, Analysis of transcriptional activation by TaDTG6-A, TaDTG6-B^Del574, TaDTG6-B^In574, and TaDTG6-D in N. benthamiana leaves. Left shows the schematic diagrams of the effector constructs; right shows relative LUC activity. Values represent means ± sd from at least three independent experiments. Statistical significance was determined by two-sided Student’s t test (**P < 0.01).

Figure 5

Screens for TaDTG6-B^Del574 and TaDTG6-B^In574 interaction with their cofactors. A, Y2H assays confirming the interactions between TaDTG6-B^Del574 and six cofactors. B, Y2H assays testing for interaction between TaDTG6-B^In574 and six cofactors of TaDTG6-B^Del574. Yeast cells were grown on SD medium –Ade –His –Leu –Trp. AD, GAL4 activation domain; BD, GAL4 DNA-binding domain. C, Firefly LCI assays to further assess TaDTG6-B^Del574 and TaDTG6-B^In574 interactions with six cofactors in N. benthamiana leaves.

Figure 6

TaDTG6-B^Del574 improves wheat drought tolerance. A, D, G, Relative expression levels of TaDTG6-B in Ubipro:TaDTG6-B^In574 OE lines (A), Ubipro:TaDTG6-B^Del574 OE lines (D), and TaDTG6-B RNAi (Ri) lines (G), as determined by RT–qPCR. Immunoblot of TaDTG6-B in WT and transgenic lines are shown below the RT–qPCR results. B, E, H, SRs in Ubipro:TaDTG6-B^In574 OE lines (B), Ubipro:TaDTG6-B^Del574 OE lines (E), and TaDTG6-B RNAi lines (H) during drought treatment and recovery. Seedlings with green and expanded viable leaves were regarded as survivors. Values are means ± sd from at least three independent experiments; statistical significance was determined by a two-sided Student’s t test (**P < 0.01). C, F, and I, Representative drought tolerance phenotypes in Ubipro:TaDTG6-B^In574 OE lines (C), Ubipro:TaDTG6-B^Del574 OE lines (F), and TaDTG6-B RNAi lines (I). Photographs were taken under well-watered conditions before drought treatment and after a 3-day period of recovery with full irrigation post drought treatment. J, Distribution of TaDTG6-B^In574 and TaDTG6-B^Del574 alleles in tetraploid wheat, hexaploid landraces, and modern cultivars.

Figure 7

Genome-wide identification of TaDTG6-B^Del574 binding sites and target genes. A, Distribution of the locations of predicted binding sites within target genes. Promoter was defined as the sequence within 2-kb upstream of the predicted transcriptional start site; terminator is the sequence within 2-kb downstream of the predicted transcription termination site; gene body consists of the 5′-UTR, exons, introns, and 3′-UTR. B, Venn diagram showing the extent of overlap between genes identified as DEGs by RNA-seq and potential TaDTG6-B targets, as determined by DAP-seq analysis. C, GO classification for DEGs with predicted TaDTG6-B^Del574 binding sites. D and E, Identification of two motifs enriched in TaDTG6-B^Del574 binding sequences using MEME software. F and G, EMSA showing that the DRE/CRT motifs ACCGAC (F) and GCCGAC (G) are required for TaDTG6-B^Del574 binding to its targets. Recombinant GST-tagged TaDTG6-B^Del574 and TaDTG6-B^In574 were used in the EMSA, and GST was used as a control.

Figure 8

TaDTG6-B^Del574 can directly induce TaPIF1 transcription. A, Schematic diagram of the TaPIF1 promoter (WT [intact] and mutated). The DRE/CRT cis-elements are indicated by black boxes. B, RT–qPCR analysis of relative TaPIF1 expression levels in the WT, Ubipro:TaDTG6-B^Del574 OE lines, and TaDTG6-B RNAi lines. C, ChIP-qPCR validation of TaDTG6-B^Del574 binding sites in the TaPIF1 promoter. The fragments used in ChIP-qPCR are indicated in (A). D, EMSA of TaDTG6-B^Del574 binding to the DRE/CRT cis-elements in the TaPIF1 promoter. Biotin-labeled probes were incubated with GST or GST-tagged TaDTG6-B^Del574. 100× and 200× unlabeled competitor fragments were added to evaluate binding specificity. E-F, TaDTG6-B increases TaPIF1 promoter activity. Nicotiana benthamiana leaves were co-infiltrated with constructs encoding either TaDTG6-B^Del574 or TaDTG6-B^In574 and either intact or mutated TaPIF1 promoter. The firefly LUC/REN ratio indicates the level of transcriptional activation of the different promoters by each respective effector construct. Values are means ± sd from at least three independent experiments; statistical significance was determined by two-sided Student’s t test (**P < 0.01).

Figure 9

TaPIF1 overexpression improves drought tolerance in transgenic wheat. A, Relative expression levels of TaPIF1 in Ubipro:TaPIF1 OE lines. B, SR in Ubipro:TaPIF1 OE lines during drought treatment and recovery. C, Representative drought tolerance phenotypes in Ubipro:TaPIF1 OE lines. Photographs were taken under well-watered conditions before drought treatment and after a 3-day recovery period with full irrigation post drought treatment. D–G, Physiological parameters of Ubipro:TaPIF1 OE and WT plants under well-watered (WW) and water-deficit (WD) conditions. D, Proline contents. E, Soluble sugar contents. F, MDA contents. G, Chlorophylls contents. Values are means ± sd from at least three independent experiments; statistical significance was determined by two-sided Student’s t test (*P < 0.05; **P < 0.01).

Figure 10

A working model for TaDTG6-B^Del574 allele-mediated drought tolerance. Drought-tolerant wheat accessions carrying TaDTG6-B^Del574 allele exhibit a stronger TaDTG6-B activity of transcriptional activation and protein interactions, and also have the ability to bind to DRE/CRT cis-elements to regulate stress-responsive gene expression, resulting in the improvement of drought tolerance. Drought-sensitive wheat accessions carrying the TaDTG6-B^In574 allele have a lower TaDTG6-B activity of transcriptional activation and protein interactions, and cannot bind to DRE/CRT cis-elements, resulting in sensitivity to water deficiency.