Fig. 3: DOF6 acts as a transcriptional repressor, directly inhibiting the expression of the GA20ox3 gene across Col-0, Sichuan, and Tibet ecotypes. | Nature Communications

Fig. 3: DOF6 acts as a transcriptional repressor, directly inhibiting the expression of the GA20ox3 gene across Col-0, Sichuan, and Tibet ecotypes.

From: Allelic variations in GA20ox3 regulate fruit length and seed germination timing for high-altitude adaptation in Arabidopsis thaliana

Fig. 3

a DOF proteins recognize the core sequence AAAG near the -418 SNP and -1,211 SNP. b A heatmap visualizes DOF family genes and GA20ox3 expression during fruit development. c RT-qPCR analysis of GA20ox3 (left) and DOF6 (right) across various stages of fruit development in the Col-0. Data are shown as means ± s.d., n = 3 independent experiments. d Fruit images of Col-0, DOF6-OE1, ga20ox3-1, dof6 and ga20ox3-1 dof6 at stage 17-B. Scale bar: 2 mm. e Statistic analysis of FL in (d). f Germination assays of different lines using freshly harvested seeds on 1/2 MS medium. g Statistical analysis of the germination rate in (f). h RT-qPCR analysis of GA20ox3 in freshly harvested seeds from Col-0 and DOF6-OE1. i Dual-luciferase assays of GA20ox3Col-0 promoter activity modulated by DOF6. j The schematic diagram illustrates the A/TAAAG-box regions used for ChIP in the GA20ox3 promoter. Fragments P1-P9 represent the regions amplified for ChIP-qPCR analysis. k The EMSA results demonstrate that DOF6 directly binds to the AAAAG-box of P8 in the GA20ox3Col-0 promoter. l The mutant site is indicated, where Sic-mutant and Tib-mutant denote the substitution of T for A and A for T, respectively, at nucleotide position -418 bp of the GA20ox3 promoter. m Dual-luciferase assays were conducted to assess the promoter activity of GA20ox3Sic, GA20ox3Tib, GA20ox3Sic-mut, and GA20ox3Tib-mut in response to modulation by DOF6. n, o EMSAs reveal variations in the binding affinity of DOF6 to the GA20ox3Sic, GA20ox3Tib, GA20ox3Sic-mut, and GA20ox3Tib-mut promoters. In e, g, data are presented as means ±s.d. (n = 24/18/10/15/15 biological replicates in e; n = 3 biological replicates in g), with different letters indicating statistically significant differences (P  < 0.05) determined by one-way ANOVA with Tukey’s multiple comparisons test. In h, i, j, m, data are shown as means ± s.d. (n = 3 biological replicates), with P values obtained using an unpaired two-tailed Student’s t-test. Source data are provided as a Source Data file.

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