Fig. 3: Indel of 899 bp in the TaDT1-A promoter confers drought stress responses and TaMYC2 works upstream of TaDT1-A.

a Schematic diagram of the TaDT1-A^hapI and TaDT1-A^hapII promoter fragments used for construction of the transient expression vector. Inserting an 899-bp fragment into the TaDT1-A^hapI promoter to generate TaDT1-A^mhapI, presented as ‘mhapI’. The mutated nucleotides in the G-Box are presented in red font in the ‘mhapII’ sequences and presented as ‘mhapII’. b Wheat drought tolerance distributions of TaDT1-A^hapI (n = 51 accessions) and TaDT1-A^hapII (n = 40 accessions). c Comparison of TaDT1-A upregulated expression in varieties (ratio of drought treatment to normal treatment) grouped by the two different haplotypes. (n = 51 for TaDT1-A^hapI; n = 40 for TaDT1-A^hapII). d Correlation analysis of the upregulated expression of TaDT1-A and wheat SR in different varieties (n = 91 accessions). e Comparison of the drought tolerance of the NILs carrying the tolerant allele hapI or the sensitive allele hapII. Bar, 3 cm. f Statistical data for the SR of the NILs exposed to drought stress (n = 3 biological replicates). g Statistical data for the fresh weight of the NILs when exposed to drought stress (n = 16 biological replicates). h Transient expression assays of different promoter fragments from two TaDT1-A genotypes and two mutant TaDT1-A genotypes. (n = 6 biological replicates). i Results from yeast one-hybrid assays showing that TaMYC2 binds to G-Boxes within the 899-bp insertion. j EMSAs showing that TaMYC2 directly binds to the 899-bp insertion and G-Box. The affinity-purified fusion protein GST-TaMYC2 was incubated with biotin-labeled probes. Two independent experiments were performed with similar results. k Drought stress tolerance assay of TaMYC2-overexpressing and WT plants. Bar, 3 cm. l Statistical analysis of the survival rates of OE and wild-type plants after drought treatment and recovery (n = 3 biological replicates). m Validation of TaMYC2 binding sites in the TaDT1-A promoter by ChIP-qPCR analysis. The top diagram shows the fragments used for ChIP-qPCR (n = 3 biological replicates). Values are presented as means ± SD. For (b, c, g, h), all data are plotted with box and whiskers plots: whiskers plot represents minimum and maximum values, and box plot represents second quartile, median and third quartile. For (b–d, f–h, l), *, **, and **** indicate significant differences at P < 0.05, P < 0.01, and P < 0.0001 using the two-sided Student’s t-test. For (m), different letters were used to denote statistically significant differences, which were determined using two-way ANOVA with Tukey’s multiple comparisons test (P  <  0.05). Source data are provided as a Source Data file.