Extended Data Fig. 8: MIR156C and FLC are direct targets of LEC2.
From: A robust mechanism for resetting juvenility during each generation in Arabidopsis
a, Leaf length/width ratio of WT, mir156ac and lec2 under LD conditions. The results were shown as mean ± sd. n ≥ 12. Dash lines represented the lack of rosette leaves in some plants caused by early flowering. The data of WT were the same as Fig. 3d. The statistical analysis is shown in Supplementary Table 5. b, Leaf shapes of WT, mir156ac and lec2 under LD conditions when flowering. Scale bar, 1 cm. c, Transcript abundance of selected genes by RNA-seq. RNA counts were normalized by DESeq2 based on size factors and were shown as mean ± sd, n = 2. d, The ChIP-PCR data of H3K27me3 for MIR156A and MIR156C gene loci in WT and lec2. The symbols are as described in Extended Data Fig. 7. Two biological replicates were performed. One representative replicate with three technical repeats is shown. The results were represented by mean ± sd. Two-tailed Student’s t test, **, p < 0.01; *, p < 0.05; ns, no significant difference. The statistical analysis is shown in Supplementary Table 1. e, Competitive EMSA showing binding of LEC2-DBD (DNA-binding domain of LEC2) to the RY motif of MIR156C promoter. The relative amount of unlabeled competitive oligonucleotide is indicated on the top. Schematic of the MIR156C genomic region and the position of the RY motif are shown in Fig. 5a. The assays without GST-LEC2-DBD protein (-) or with mutated probes (mut) were performed as controls. The probe sequences are listed in Supplementary Table 3. f, ATAC-seq tracks and LEC2 ChIP data for MIR156B gene locus in WT and lec2. The ChIP-seq data of p35S:LEC2-3xFLAG-GR seedlings and ChIP-PCR data of pLEC2:LEC2-3xFLAG showed similar results. The symbols are as described in Extended Data Fig. 7. The results were represented by mean ± sem, n = 3. Two-tailed Student’s t test, *, p < 0.05; ns, no significant difference. The statistical analysis is shown in Supplementary Table 1. See also Fig. 6h. g, ATAC-seq tracks and p35S:LEC2-3xFLAG-GR ChIP-seq data for MIR157A and MIR157C and FLC gene loci. Symbols were described in Extended Data Fig. 7. See also Fig. 6h. h, Expression levels of MIR156B and miR156 in the embryos (3 days after pollination) of WT and rkd4 mutant. n = 3 independent biological replicates. The results are shown as mean ± sem. Two-tailed Student’s t test, **, p < 0.01. The statistical analysis is shown in Supplementary Table 1. i, Induction of MIR156B and miR156 by RKD4. We generated an inducible line for RKD4 based on the GR-DEX system. The 10-day-old p35S:RKD4-GR seedlings were treated with DMSO (mock) or 10 μM DEX for 4 h. n = 3 independent biological replicates. The results are shown as mean ± sem. Two-tailed Student’s t test, **, p < 0.01; ****, p < 0.0001. The statistical analysis is shown in Supplementary Table 1.
