oi’ cell expansion is repressed by FIS PcG proteins and induced by endosperm growth.

<p><b>(A)</b> Average length of the most proximal oi’ cell as observed in longitudinal sections of wild type and mutant seeds imaged using the mPS-PI technique. Asterisks indicate statistical difference (two-tailed student's <i>t</i> test, P < 0.05). Error bars: standard deviations. n>20. <b>(B-C)</b> Longitudinal sections of <i>kpl-1</i> only-endosperm (B) and only-embryo (C) seeds at 6 DAF imaged using the mPS-PI technique. Ecotype Ws. <b>(D)</b> GFP fluorescence image of a <i>ProFIE</i>:<i>gFIE-GFP</i> ovule at stage 3-VI. oi1 and oi2 cell layers are marked by white lines whereas oi’ cells are marked by a red line. <b>(E-F)</b> Longitudinal sections of <i>fie12/+</i> (E) and <i>msi1-1/+</i> (F) unfertilized large autonomous seeds at 6 DAF imaged using the mPS-PI technique. Ecotype Col. <b>(G)</b> Model for the development of the seed oi’. Black arrows indicate functional relationships. Scale bars, 50 μm. oi’ and chalazal sub-epidermal cells are highlighted in red.</p

Developmental patterning of sub-epidermal cells in the outer integument of Arabidopsis seeds

<div><p>The seed, the reproductive unit of angiosperms, is generally protected by the seed coat. The seed coat is made of one or two integuments, each comprising two epidermal cells layers and, in some cases, extra sub-epidermal cell layers. The thickness of the seed-coat affects several aspects of seed biology such as dormancy, germination and mortality. In Arabidopsis, the inner integument displays one or two sub-epidermal cell layers that originate from periclinal cell divisions of the innermost epidermal cell layer. By contrast, the outer integument was considered to be two-cell layered. Here, we show that sub-epidermal chalazal cells grow in between the epidermal outer integument cell layers to create an incomplete three-cell layered outer integument. We found that the MADS box transcription factor TRANSPARENT TESTA 16 represses growth of the chalaza and formation of sub-epidermal outer integument cells. Finally, we demonstrate that sub-epidermal cells of the outer and inner integument respond differently to the repressive mechanism mediated by FERTILIZATION INDEPENDENT SEED Polycomb group proteins and to fertilization signals. Our data suggest that integument cell origin rather than sub-epidermal cell position underlies different responses to fertilization.</p></div

oi’ formation.

<p><b>(A-B)</b> Longitudinal sections of wild type ovules at stage 2-III (A) and 3-VI (B) imaged using the mPS-PI technique. Ecotype Col. ii primordium, oi primordium and chalaza are highlighted in green, blue, and red, respectively. Highlighting of the chalazal tissue in red is inferred by the position of the integument primordia. <b>(C-D)</b> Longitudinal sections of wild type mature ovules imaged using the mPS-PI technique. Ecotype Col. oi’ is highlighted in red. <b>(E-F)</b> Longitudinal sections of wild type seeds at 6 DAF imaged using the mPS-PI technique. Ecotype Col. oi’ is highlighted in red. <b>(G-H)</b> Three-dimensional longitudinal (G) and transverse (H) sections of a wild type seed at 6 DAF imaged using the mPS-PI technique. Ecotype Ws. oi’ is highlighted in red. Scale bars, 10 μm (A) and 50 μm (B-H).</p

Growth of the chalazal tissue promotes oi’ formation.

<p><b>(A-C)</b> Longitudinal sections of <i>tt16-1</i> (A), wild type (B), and <i>tt16-3</i> (C) seeds at 6 DAF imaged using the mPS-PI technique. Ecotype Ws. oi’, nucellus, and proximal oi are highlighted in red, orange and yellow, respectively. Red arrows indicate the chalaza height. <b>(D)</b> Average chalazal height as observed in central longitudinal sections of wild type and mutant seeds imaged using the mPS-PI technique. Chalaza height was measured from the proximal nucellus till the proximal oi as shown in panels A and B (red arrows). Asterisks indicate statistical difference between wild type and mutant lines (two-tailed student's <i>t</i> test, P < 0.05). Error bars: standard deviations. n>20.</p

Analysis of <i>MYB115</i>, <i>AAD2</i>, and <i>AAD3</i> transcript levels by quantitative RT-PCR in <i>ProAT2S2</i>:<i>MYB115</i> embryos.

<p>The four independent transformants considered, TAR3, TXR2, TGR5, and TJR2 (in order from left to right), are displayed in the same order in the three graphs in the figure. RT-qPCR analysis of transcript abundance in cDNA prepared from excised embryos aged 14 DAA was carried out to assess efficient overexpression of the transgene (<i>MYB115</i>) and of its targets (<i>AAD2</i> and <i>AAD3</i>). Values are the means and SE of three replicates performed on cDNA dilutions obtained from three independent mRNA extractions. Asterisks indicate significant differences from the wild type according to <i>t</i>-test at *** P<0.001, **P<0.01, and *P<0.05, respectively.</p

Increased omega-7 fatty acid content of <i>ProAT2S2</i>:<i>MYB115</i> seeds.

<p>The five independent transformants considered, TCR4, TAR3, TXR2, TGR5, and TJR2 (in order from left to right), are displayed in the same order in the four graphs in the figure. (A) RT-qPCR analysis of transcript abundance in cDNA prepared from maturing seeds aged 14 DAA to assess efficient overexpression of the transgene. Values are the means and SE of three to 12 replicates performed on cDNA dilutions obtained from three independent mRNA extractions. (B-D) Relative proportion of ω-7 fatty acids (<i>cis</i>-ω-7 C16:1, <i>cis</i>-ω-7 C18:1, and <i>cis</i>-ω-7 C20:1) in mature dry seeds (B), in embryos (C), and in endosperm fractions (D) dissected from mature seeds. Values are the means and SE of five replicates performed on batches of 20 seeds from five plants. Asterisks indicate significant differences from the wild type according to <i>t</i>-test at *** P<0.001 and **P<0.01, respectively.</p

Validation of the <i>AT2S2</i> promoter sequence.

<p>(A-C) Analysis of relative mRNA accumulation of <i>AT2S2</i> was performed in different plant organs (A), in developing seeds (B), and in developmental series of endosperm (Endo.) and embryo fractions (C). The results obtained were standardized to the <i>EF1αA4</i> (<i>EF</i>) gene expression level. Values are the means and SE of three to six replicates carried out on cDNA dilutions obtained from three independent mRNA extractions. DAA, days after anthesis; Fl, flowers; Rl, rosette leaves; Ro, roots; St, stems. (D-J) Pattern of activity of the <i>ProAT2S2</i>:<i>uidA</i> cassette in maturing embryos harvested 10 (D), 12 (E), 14 (F), or 16 days after anthesis (DAA) (G) and in endosperm fractions harvested 14 (H) or 16 DAA (I). A close-up of a peeled endosperm layer aged 14 DAA is presented in (J). For histochemical detection of GUS activity, tissues were incubated 4 hours in a buffer containing 2 mM each of potassium ferrocyanide and potassium ferricyanide. Microscopy observations were performed using Nomarski optics. Bars = 100 μm in (D-I), 10 μm in (J).</p

Comparison of the omega-7 fatty acid content of seeds from engineered lines of <i>A</i>. <i>thaliana</i> obtained by different groups.

<p>Comparison of the omega-7 fatty acid content of seeds from engineered lines of <i>A</i>. <i>thaliana</i> obtained by different groups.</p

Characterization of transgenic seeds overexpressing <i>MYB115</i>.

<p>The five independent transformants considered, TCR4, TAR3, TXR2, TGR5, and TJR2 (in order from left to right), are displayed in the same order in every graph in the figure. (A) Mature seed dry weight. Values are the means and SE of five replicates carried out on batches of 20 seeds from five plants. (B) Mature seed length. Values are the means and SE of 100 measurements carried out on seeds from five plants. (C) Mature seed width. Values are the means and SE of 100 measurements carried out on seeds from five plants. (D, E) Total fatty acid content of mature dry seeds, expressed in μg.mg^-1 DW (D) or in μg per seed (E). Values are the means and SE of five replicates carried out on batches of 20 seeds from five plants. (F) Total seed fatty acid content of embryos dissected from mature dry seeds. Values are the means and SE of five replicates carried out on batches of 20 embryos from five plants. (G) Total seed fatty acid content of endosperm fractions dissected from mature dry seeds. Values are the means and SE of five replicates carried out on batches of 20 endosperm fractions from five plants. Asterisks indicate significant differences from the wild type according to <i>t</i>-test at *** P<0.001, **P<0.01, and *P<0.05, respectively.</p

MOESM1 of Combining laser-assisted microdissection (LAM) and RNA-seq allows to perform a comprehensive transcriptomic analysis of epidermal cells of Arabidopsis embryo

Additional file 1: Fig. S1. Principal Components Analysis (PCA) on normalized counts of the samples of the pilot experiment (RNA quantity from 5 ng to 10 pg). First and second components are shown, along with the percentage of variance explained