Fig. 4: Altered DNA methylation in 3′ terminal region of HB21 affects its expression.

a structure of HB21. The exons are shown as black boxes, while the introns are shown as black lines. The promoter is shown as grey box. Eight probes were designed to detect cytosine methylation on HB21. Lower, diagram of the different truncations of the HB21 gene fused with GFP. b cytosine methylation pattern of HB21 in wild-type and efd buds. The red bar includes the methylation analyses of the cytosines in 1461–1476 bp. c RT-qPCR expression analyses of the truncations of HB21 in tobacco leaves. Data are presented as mean values ±SD. The error bars represent the SDs from three independent biological repeats. Student’s t test was used for statistical analyses of the difference between the wild type and mutants, **p < 0.01, ***p < 0.001 (p-values are shown in the figure). d GFP signal of the four transgenic constructs with truncations of gHB21 in tobacco leaves. Bars = 40 μm. Three different leaves for each transgenic constructs were selected to be analysed. e diagram of the cytosines in the 1435–1540 region. The methylated cytosines were lost in the efd mutant. The cytosine pattern was shown as the circles (CG), rectangles (CHG) and triangles (CHH) as shown at the top row of HB21 geno. The methylated cytosines are shown as the full circles, rectangles and triangles, while the unmethylated cytosines are shown as empty circles, rectangles and triangles. The red frame indicates that the cytosines in this region were unmethylated in efd. f the genomic sequence and the point mutation in the 1458–1478 region. There are four cytosines: 1461, 1464, 1467 and 1476. g RT-qPCR results of the point mutated HB21 transgenic tobacco leaves. h the GFP signal detection of the point mutations. Data are presented as mean values ±SD. The error bars represent the SDs from three independent biological repeats. Student’s t test was used for statistical analysis. ns, no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001 (p-values are shown in the figure). Bars = 40 μm. i EMSA image of the potential HB21 probe (unmethylated probe) incubated with the EFD protein.