Fig. 5: HAN directly represses CDF4 transcription.

a,b, DEX induction of 35S:HAN-GR causes repression of the pCDF4:NLS-3xYFP reporter in the CCs (a), whereas DEX alone has no effect (b). Representative images of the indicated genotypes from 3 independent biological replicates. c, Relative expression of CDF4 in Col-0 and 35S:HAN-GR after induction with DEX at the indicated timepoints. Data are means ± s.d. of 5 independent biological replicates. ****P < 0.0001 using one-way ANOVA with Dunnett’s multiple comparisons test. d, Representative confocal images showing expression of pCDF4:NLS-3xYFP in Col-0 wild-type and han-30 background. Dashed white lines indicate QC. e, Quantification of YFP intensity in two central QC cells of each genotype. Data are means of n > 10 roots of each genotype. ***P = 0.000178 using a two-sided Student’s t-test. f, RT–qPCR expression of CDF4 in Col-0 wild type and 35S:HAN-GR after treatment with Mock, DEX (10 µM), CHX (10 µM) and DEX + CHX (10 µM) for 4 h. Data are means ± s.d. of 4 independent biological replicates. ^NSP = 0.113 using a two-sided Student’s t-test. g,h, ChIP analysis. g, Diagram showing the 3-kb-long promoter region of CDF4 upstream of the ATG and positions of the ChIP–qPCR primers. Magnification depicts 1 kb of the CDF4 promoter containing the predicted GATA-binding motifs for HAN and the WOX5 binding site (ref. ¹⁴) for comparison. h, Specific HAN-3xFlag enrichment as % input at the P1 and P2 sites upon 6 h DEX induction (10 µM) of 35S:Gal4-VP16-GR; UAS:3xFLAG-HAN and the Col-0 wild type. GAPC2 was used as a negative control. Data are means ± s.d. of 3 independent biological replicates. ^NSP_P3 = 0.533, **P_P2 = 0.00470, *P_P1 = 0.0135, ^NSP_GAPC2 = 0.147 using a two-sided Student’s t-test. i, RT–qPCR expression of CDF4 in the Col-0 wild-type, 35S:WOX5-GR and 35S:WOX5-GR han-30 roots after treatment with DEX for 4 h. Data are means ± s.d. of 4 independent biological replicates. ****P_{Col-0 vs 35S:WOX5-GR} < 0.0001, *P_{Col-0 vs 35S:WOX5-GR han-30} = 0.0129, ***P_{35S:WOX5-GR vs 35S:WOX5-GR han-30} = 0.0008 using one-way ANOVA with Tukey’s multiple comparisons test. Scale bars, 20 μm.