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Similarity between soybean and Arabidopsis seed methylomes and loss of non-CG methylation does not affect seed development

  1. Robert B. Goldberga,5
  1. aDepartment of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA 90095;
  2. bDepartment of Plant and Microbial Biology, University of California, Berkeley, CA 94720;
  3. cSection of Plant Biology, Division of Biological Sciences, University of California, Davis, CA 95616
  1. Contributed by Robert B. Goldberg, October 3, 2017 (sent for review September 25, 2017; reviewed by Z. Jeffrey Chen, Brian A. Larkins, and Lila O. Vodkin)

  1. Fig. 2.

    Genome-wide methylation changes during soybean seed development and germination. DNA methylation levels in 500-kb windows across the genome are represented as box plots (A) and chromosome heat maps (B). The highest methylation levels (%) for heat maps tracks are 96, 80, and 9 for CG, CHG, and CHH contexts, respectively. Gene and TE tracks represent gene and TE densities in 500-kb windows across the genome. Gm, Glycine max. See Table 1 for abbreviations.

  2. Fig. 3.

    Comparison of methylomes between soybean seed parts and SC layers. (A) Paraffin sections of cot-stage SC, embryonic AX, and embryonic COTL before and after capture by LCM. (B) Whole-mount pictures of SC, AX, and COTL from em- and mm-stage EMB and seeds. (C) Box plots of DNA methylation levels in 500-kb windows across the genome in different seed parts. Asterisks indicate significant comparisons between SC and other seed parts at the same stage (t test, P < 0.001 and fold change > 1.5). (D) Paraffin cross-section of an em-stage seed (Upper), and a plastic section of SC layers (Lower), which is the red boxed region shown in the whole-seed section. (E) Paraffin cross-sections of em-stage SC-PA and SC-PY layers before and after capture by LCM. (F) Box plots of LCM-captured em-stage COTL, SC, SC-PA, and SC-PY DNA methylation levels in 500-kb windows across the genome. Asterisks indicate significant comparisons between SC, SC layers, and the COTL (t test, P < 0.001 and fold change > 1.5). (G) Box plots of TE siRNA levels (reads per million mapped reads) in em-stage SC, AX, and COTL. Asterisks indicate statistically significant comparisons between SC and other seed parts (t test, P < 0.001 and fold change > 1.5). [Scale bars, 100 μm (A, D, and E) and 1 mm (B).] See Table 1 for abbreviations.

  3. Fig. 4.

    DNA methylation levels in endoreduplicated and nonendoreduplicated soybean COTL regions. (A) Paraffin cross-sections of em-stage COTL endoreduplicated ABPY and nonendoreduplicated ADPY before and after capture by LCM. (Scale bars, 100 μm.) (B) Box plots of DNA methylation levels in 500-kb windows across the genome in em-stage COTL ABPY and ADPY regions. (C) Log2 ratios of normalized DNA reads in 500-kb windows across all 20 chromosomes from: (i) em-stage ABPY and ADPY COTL regions, and (ii) em and mm seed parts (COTL and AX). See Table 1 for abbreviations.

  4. Fig. 5.

    Methylation levels and mRNA accumulation patterns of major soybean seed-specific gene classes during seed development and germination. (A) mRNA accumulation patterns. RPKM represents reads per kilobase per million sequences, and were taken from the Goldberg-Harada soybean (i) whole seed RNA-Seq dataset, GEO accession no. GSE29163 (37), and (ii) COTL-specific RNA-Seq dataset GSE29134 (sdlg-COTL). (B) Methylation levels of CG-, CHG-, and CHH-context sites are shown in genome browser view (vertical lines). Gene structures, transcription directions (arrows), and TEs are shown below each genome browser view. Adjacent genes are not shown. The size of each genomic region, including 2 kb of 5′ and 3′ flanking regions, is shown at the bottom. GmABI3-1, abscisic acid insensitive3-1; GmCG-1,β-conglycinin-1; GmFAB2C, stearoyl-ACP desaturase 2C; GmFUS3-2, FUSCA 3-2; GmGy1, glycinin 1; GmKTi3, Kunitz trypsin inhibitor 3; GmLEC1-1, Leafy Cotyledon 1-1; GmLe1, lectin 1. See Table 1 for developmental stage abbreviations. Gm, Glycine max.

  5. Fig. 6.

    Genome-wide methylation changes during Arabidopsis seed development. (A) Arabidopsis seed stages and major developmental events. Adapted from ref. 38). Seed and EMB images are not drawn to scale. Brackets indicate stages investigated. Box plots (B) and chromosome heat maps (C) of DNA methylation levels in 100-kb windows across the genome. The highest methylation levels were 96%, 80%, and 10% for CG, CHG, and CHH contexts, respectively. Gene and TE tracks represent densities of genes and TEs in 100-kb windows along the genome. (D) Box plots of DNA methylation levels in 100-kb windows across the genome in mg-stage EMB and SC. (Scale bars, 0.1 mm.) Asterisk indicates a significant comparison between EMB and SC (t test, P < 0.001 and fold change > 1.5). mRNA accumulation patterns (E) and genome browser views of DNA methylation levels (F) for major seed-specific gene classes. Transcript signal intensities were obtained from microarray analysis (38). Methylation levels of CG-, CHG-, and CHH-context sites are shown in genome browser view (vertical lines). Gene structures, transcription directions (arrows) and TEs are shown below each genome browser view. Adjacent genes are not shown. The size of each genomic region, including 2 kb of 5′ and 3′ flanking regions, is shown at the bottom. AtCRA1, Cruciferin 1. Names of other genes are defined in the legend to Fig. 5. At, Arabidopsis thaliana. See Table 1 for abbreviations of seed stages.

  6. Fig. 7.

    Comparison between Arabidopsis wild-type and ddcc seed development and germination. (A) Box plots of methylation levels in 100-kb windows across the wild-type and ddcc genomes. (B) Nomarski photographs of wild-type and ddcc seeds at different developmental stages. (Scale bars, 50 μm.) (C) DAPI-stained nuclei of pmg-COTL and leaves. (Scale bars, 5 μm.) Comparison of nuclear sizes (110 nuclei) (D), 4-d sdlg morphologies (E), and germination percentages (F) for wild-type and ddcc seeds. Five replicates with 50 seeds each were used in the germination assays.

  7. Fig. 8.

    Comparison between Arabidopsis wild-type and ddcc pmg seed transcriptomes. (A) Correlation between wild-type and ddcc seed mRNA accumulation levels. (B) Differentially expressed genes in ddcc seeds. DOWN, down-regulated; UP, up-regulated. (C) Genome browser view of major seed-specific mRNA accumulation patterns in ddcc and wild-type seeds. Arrows show the transcription directions. Gene names are defined in the legends to Figs. 5 and 6. (D) Methylation status of 5′ flanking 1-kb region of differentially expressed genes.

  8. Fig. 9.

    TE transcriptional activity in Arabidopsis wild-type and ddcc pmg seeds. (A) Correlation between wild-type and ddcc seed TE RNA accumulations levels. Red and blue dots represent de-repressed and up-regulated TEs, respectively. (B) Box plots comparing de-repressed and up-regulated TE RNA accumulation levels in wild-type and ddcc seeds. (C) Genome browser view of the methylation pattern and RNA accumulation profile of a de-repressed Copia TE in pmg wild-type and ddcc seeds. LTR, long terminal repeat. (D) Major protein classes involved in TE transposition encoded by 106 de-repressed and up-regulated ddcc seed TE RNAs (SI Materials and Methods). (E) TE copy number changes between ddcc and wild-type seeds. Box plots show log2 ratios of normalized read depths from ddcc versus wild-type TEs. The gene control includes all genes in the Arabidopsis genome. (F) Methylation levels across 106 de-repressed and up-regulated TEs in wild-type seeds. The no RNA control used in E and F represent 106 randomly selected TEs which have (i) no detectable RNA wild-type reads and (ii) similar TE family distribution and lengths compared with the 106 de-repressed and up-regulated TEs. (G) Methylation levels across 106 de-repressed and up-regulated TEs in Ws-0 wild-type seeds during Arabidopsis seed development.

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