Fig. 5: MADS31 maintains inner nucellus identity by repressing NRPD4b expression in the ovule.
a, Relative expression level (RT–qPCR) of NRPD4b in WT and mads31. Data are shown as mean ± s.d.; n = 3 replicates; two-sided t-test. b, In situ hybridization of NRPD4b in WT and mads31 ovules. The black dotted lines indicate ovules. Sense probe serves as negative control. Scale bars, 50 μm. c, The genomic region of NRPD4b including promoter and coding region. CArG motifs and restriction enzyme sites are marked. d, Normalized luciferase activity (LUC/REN) regulated by NRPD4b promoter in the presence of MADS31 or empty vector (EV, negative control). Data are shown as mean ± s.d.; n = 8 replicates; two-sided t-test. e, Four DNA fragments with CArG motif and one without CArG motif tested by ChIP–PCR. No antibody (ab) serves as negative control. Data are shown as mean ± s.d.; n = 3 replicates; two-sided t-test. f, Chop–PCR assay of DNA methylation in NRPD4b promoter in WT and mads31. Experiments were repeated 3 times, with similar results. g, Overexpression of NRPD4b decreases seed set rate. Top: relative expression level of NRPD4b in transgenic lines, wild-type and mads31 pistils at Ov7/8 stages. Data are shown as mean ± s.d.; n = 3 replicates. Bottom: seeds set rates of transgenic, wild-type and mads31 plants. Data are shown as mean ± s.d.; n = 4 spikes; two-sided t-test for unpaired two-sample data. h, Early (Ov2 and Ov3) and mature (Ov10) stages of wild-type and Ubi::NRPD4b ovules. For Ubi::NRPD4b, Left: toluidine blue-stained longitudinal sections. Middle: LM19-labelled demethylesterified pectin in the cell walls of inner nucellus. Right: diagrams of cell arrangement in the nucellus. Green, germ cell; yellow, inner nucellus. Ratios of area of inner nucellus versus outer nucellus are shown as mean ± s.d.; n = 5 ovules; two-sided t-test for unpaired two-sample data. Scale bars, 25 μm. All experiments were repeated at least 3 times, with similar results.
