Fig 1.
Natural variation in imprinting is associated with differences in HDG3 expression levels.
(A) HDG3 expression is decreased in Col x Cvi endosperm compared to Col, as determined by mRNA-seq. (B) Schematic of the HDG3 locus. DMR, differentially methylated region. (C) In situ hybridization of HDG3 (purple) in F1 seeds from the indicated crosses. Female parent is written first. In crosses where Col is the male parent, HDG3 is detected in the micropylar (MCE), peripheral (PEN), and chalazal (CZE) endosperm. Arrowheads indicate nuclear-cytoplasmic domains in the PEN. Number of seeds with shown pattern out of total seeds assayed is in corner of each image. Scale bars, 50 μm. (D) RT-qPCR analysis of relative HDG3 transcript abundance in F1 endosperm. Values are the average of 3 biological replicates, bars represent upper and lower range. (E) Schematic representation of relationship between HDG3 methylation, expression, and imprinting in endosperm. Thickness of arrows denotes relative expression level. Lollipops represent methylated (filled) and unmethylated (open) cytosines.
Fig 2.
Phenotypic effects of mutation of HDG3 in Col.
(A) In situ hybridization of HDG3 (purple) in seeds from the indicated crosses. Female parent is written first. Scale bars, 50 μm. (B) Endosperm cellularization is slightly delayed in hdg3 compared to wild-type at 5 DAP. For each seed the embryo stage was determined and then the stage of endosperm cellularization was defined as normal, early, or delayed given that embryo stage. (C) Seed weight of wild-type and hdg3 seeds. Individual data points and mean +/- SD shown. P-value from unpaired two-tailed t-test. (D) Seed area is significantly reduced in hdg3 seed (n = 275) compared to wild-type siblings (n = 376) (p = 8.51e-11 by Welch’s two tailed t-test). Seeds were quantified with ImageJ.
Fig 3.
Transcriptional effects associated with low HDG3 expression.
(left panel) Genes downregulated in hdg3 mutant endosperm also have reduced expression in Col x Cvi endosperm compared to Col. The plot shows the expression profile of genes with significantly altered expression in hdg3-1 endosperm (p<0.05). Genes were hierarchically clustered by Euclidean distance and complete linkage using Gene-E. (right panel) A subset of putative developmental regulators with reduced expression in hdg3 and Col x Cvi endosperm.
Fig 4.
Gain of Cvi HDG3 paternal allele methylation in endosperm.
Total methylcytosine 5’ of HDG3 in F1 endosperm, determined by bisulfite-PCR. Maternally inherited Col allele in orange, paternally inherited Cvi allele in blue. Scale to 100%, tick marks below line indicate unmethylated cytosines. Col x Cvi data published in [20].
Fig 5.
HDG3 is imprinted in Cvi HDG3 IR lines.
(A) HDG3 in situ hybridization for indicated genotypes. Arrowheads indicate regions of in situ signal. Right panels shows magnification of chalazal region. Scale bars, 50 μm. (B) RT-qPCR of relative HDG3 transcript abundance in F1 endosperm at 6–7 DAP. Dashed line separates experiments done at different times. Left, avg of 3 technical replicates. Right, avg of biological duplicates. Bars show upper and lower range. (C) % of HDG3 from Cvi allele in endosperm by TaqMan RT-qPCR assay.
Fig 6.
Effects of HDG3 imprinting on Cvi seed development.
(A) Aniline blue and safranin O staining of seed sections at 5 DAP from the indicated F1 seeds. Scale bars, 50 μm. (B) Phenotypic characterization of sectioned seeds, assaying degree of endosperm cellularization relative to embryo stage. (C) Seed weight in selfed Cvi and Cvi HDG3 IR plants. Individual data points and mean +/- SD shown. P-value from unpaired two tailed t-test. (D) Seed area for self-fertilized Cvi (n = 287 seeds), Cvi HDG3 IR 2–5 (n = 496) and Cvi HDG3 IR 3–4 (n = 386). Differences between IR seeds and Cvi are significant at p < 2.2e-16 as determined by Welch’s two-tailed t-test.
Fig 7.
Schematic summary of relative seed development at 5 DAP.
Shapes represent phenotypic space occupied by the indicated genotypes.