Research ArticlePLANT SCIENCES

Pollen tube contents initiate ovule enlargement and enhance seed coat development without fertilization

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Science Advances  28 Oct 2016:
Vol. 2, no. 10, e1600554
DOI: 10.1126/sciadv.1600554
  • Fig. 1 Transcriptome analysis for ovules with or without fertilization.

    (A) Up-regulated genes in ovules crossed with WT (WT) and gcs1/gcs1 (gcs1) pollen compared with those in ovules without pollination (NPT) at 12, 24, and 48 HAP. Number of up-regulated genes for each sample fraction and that of common up-regulated genes in WT (red circle; WT > NPT) and gcs1 (green circle; gcs1 > NPT) are indicated. Numbers of overlapping up-regulated genes are indicated in orange. (B) Cluster analysis of the tested sample fractions. Bar indicates the height of branches. gcs1_12HAP and WT_12HAP are highlighted in pink, and gcs1_24HAP and WT_24HAP are highlighted in blue, showing the similarity between clusters. (C) Early-response genes triggered by PTC release were screened in the two classes indicated and pooled for further expression analyses in (D) (see the Supplementary Materials for details). (D) Temporal expression of the identified genes in NPT, WT, and gcs1. Expression peaks were at 12 HAP in WT and gcs1. (E) Temporal expression of the genes for cell expansion (expansin), cell division (cyclin), and seed coat development in NPT, WT, and gcs1.

  • Fig. 2 Discovery of POEM.

    (A and B) POEM after crossing WT pistils (♀) with hap2-1/+ pollen (♂). (a) Largest fertilized ovule with a pollen tube (pt). (b) Smallest ovule without a pollen tube. (c) Intermediate ovule with a pollen tube. (C to E) Ovule integument cells observed at 2 DAP using an RPS5Apro::tdTomato-LTI6b marker line. (C) Largest cells (WT) after crossing +/+ pollen, indicating that all the ovules were fertilized. (D) Small cells without pollen tube (Pt–). (E) Intermediate cells with gcs1/gcs1 pollen tube(s) (gcs1), indicating that almost all the ovules were unfertilized. (F to H) Yellow fluorescent protein (YFP) spots using a CycB1;2pro:CycB1;2::NLS-YFP cell division marker line after crossing with WT (F), no pollen (G), and gcs1/gcs1 (H). (I) Number of spots (±SD) observed: 0 DAP, 11.2 ± 1.7 (all); 1 DAP: 25.9 ± 2.9 (W, wild type), 12.5 ± 2.2 (g, gcs1), and 5.3 ± 2.3 (N, no pollen); 2 DAP: 10.1 ± 2.7 (W), 0.9 ± 0.9 (g), and 3.1 ± 1.7 (N); 3 DAP: 5.4 ± 1.7 (W), 0.1 ± 0.2 (g), and 1.1 ± 1.2 (N). (J to L) Ovules at 3 DAP, stained by vanillin in WT (J); unstained virgin ovules (K); partially stained ovules, which were fertilized with gcs1/gcs1 pollen (L). v, vanillin-stained zone. Scale bars, 100 μm (A and J to L); 60 μm (C to E); 40 μm (F to H).

  • Fig. 3 PTC triggers POEM.

    (A and B) PTC release ratio at 1 DAP following crossing with lre/lre pistil and LAT52pro::GFP pollen (PTC marker): (A) 49.5 ± 10.8% (n = 12) of ovules without and (B) 50.5 ± 10.8% of ovules with PTC release. (C to E) Confocal microscopy images after aniline blue staining and the POEM ratio in each ovule at 2 DAP following crossing lre/lre ovules with gcs1/gcs1 pollen. (C) No pollen tube control. (D) Ovule with pollen tube(s) but no POEM (46.9 ± 11.8%; n = 13). (E) Ovule with pollen tube(s) and POEM (53.1 ± 11.8%; n = 13). (F to H) Survival ratio of synergid cell(s) at 2 DAP following crossing with lre/lre pistil with FGR8.0 background and gcs1/gcs1 pollen. (F) Both synergid cells (84.6%) broken. (G) One synergid cell survived (12.5%). (H) Both synergid cells survived (3.0%) in each ovule. (I) Bar graph indicating ratios with pollen tube burst (50.5%), POEM (gcs1/gcs1; 53.1%), POEM (WT; 49.1%), and synergid cell (SY) death (97.1%). (J and K) Schematic diagrams of POEM. Pollen tube entry into an ovule is not sufficient to trigger POEM (J), but PTC release triggers POEM (K). Scale bars, 50 μm (A to H).

  • Fig. 4 PTCs increase the number of central cell/autonomous endosperm nuclei.

    (A to D) WT (C24) virgin ovules with a central cell at 3 DAE (days after emasculation) (100%) (A) and 4 DAE (97.7%) (B). gcs1/gcs1-pollinated ovules with a central cell nucleus (92.2%) (C) and with two nuclei (6.8%) (D) at 2 DAP. (E to H) A central cell nucleus (96.4%) (E) and two nuclei (3.6%) (F) in a mea/mea ovule at 3 DAE. gcs1/gcs1-pollinated mea/mea ovule with two nuclei (16.4%) (G) and four nuclei (6.3%) (H) at 2 DAP. (I) Ratios of the number of central cell/endosperm nuclei. Pollen tube insertion (P+) increased the nuclei ratios (≥2) for no insertion (P−) from 0 to 7.8% (WT, 2 DAP), 2.3 to 12.8% (WT, 3 DAP), 3.6 to 22.6% (mea/mea, 2 DAP), and 6.0 to 51.7% (mea/mea, 3 DAP). Colors indicate number of nuclei. (J and K) Vanillin staining of gcs1/gcs1-pollinated WT (J) and mea/mea (K) pistils at 3 DAP. (J) Few ovules stained. (K) About 50% stained (right) but not without pollination (left). (L) Nonpollinated (left) and gcs1/gcs1-pollinated (right) mea/mea pistils at 3 DAP. (M) PTC induces ovule enlargement, seed coat development, and central cell/endosperm proliferation. Scale bars, 50 μm (A to H); 1 mm (J to L).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/10/e1600554/DC1

    fig. S1. Steps to identify PTC-induced genes.

    fig. S2. Expansion of ovule integument cells by pollen tube insertion.

    fig. S3. Enlargement of ovules by pollen tube insertion.

    fig. S4. Fertilization is not required for POEM.

    fig. S5. Expression of the embryo- and endosperm-specific marker genes was absent in the enlarged ovules after fertilization failure.

    table S1. All RNA-Seq analysis data on the ovules pollinated with NPT, WT, and gcs1/gcs1.

    table S2. List of genes in Fig. 1A.

    table S3. Data for early-response genes in cases of Zero_HAP = 0.

    table S4. Data for early-response genes in cases of Zero_HAP > 0.

    table S5. Confirmation data for early-response genes using unfertilized ovules.

    table S6. Spatial expression patterns in the ovules for the early-response genes listed in tables S3 and S4.

    table S7. Data for the genes associated with embryo and endosperm development, cell expansion, cell division, and seed coat development as well as internal references.

    References (3134)

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Steps to identify PTC-induced genes.
    • fig. S2. Expansion of ovule integument cells by pollen tube insertion.
    • fig. S3. Enlargement of ovules by pollen tube insertion.
    • fig. S4. Fertilization is not required for POEM.
    • fig. S5. Expression of the embryo- and endosperm-specific marker genes was absent in the enlarged ovules after fertilization failure.
    • References (3134)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • table S1 (Microsoft Excel format). All RNA-Seq analysis data on the ovules pollinated with NPT, WT, and gcs1/gcs1.
    • table S2 (Microsoft Excel format). List of genes in Fig. 1A.
    • table S3 (Microsoft Excel format). Data for early-response genes in cases of Zero_HAP = 0.
    • table S4 (Microsoft Excel format). Data for early-response genes in cases of Zero_HAP > 0.
    • table S5 (Microsoft Excel format). Confirmation data for early-response genes using unfertilized ovules.
    • table S6 (Microsoft Excel format). Spatial expression patterns in the ovules for the early-response genes listed in tables S3 and S4.
    • table S7 (Microsoft Excel format). Data for the genes associated with embryo and endosperm development, cell expansion, cell division, and seed coat development as well as internal references.

    Download tables S1 to S7

    Files in this Data Supplement:

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