Model for FMB regulation by GUN1-dependent retrograde signal.

<p>Most leaf cells express <i>FIL</i> and have miR165/166 activity just after leaf initiation. However, during the early developmental stages, the <i>FIL</i>-expressing and miR165/166-active cells switch the nuclear gene expression state to that expressing <i>PHB</i>-like genes, thus FMB shifts. When plastid gene expression machinery is functional (A), the gene expression switch in nuclei progress smoothly regardless whether GUN1 is functional or not. The pace of this gene expression switch is important for the full lamina expansion. When the plastid gene expression machinery is impaired (B), the <i>GUN1</i>-dependent retrograde signal affects the nuclei to delay or stop the gene expression switch. This plastid effect contributes to prevent the wide lamina expansion. Possibly, the <i>GUN1</i>-dependent retrograde signal regulates also other nuclear genes to repair the plastid condition. When the plastid gene expression machinery is impaired and the plant is devoid of the <i>GUN1</i>-dependent retrograde signal (C), the switch in nuclear gene expression progress normally and lamina expands despite the absence of photosynthetic activity.</p

A simple mutual repression of genes mediated by mobile factors easily shifts the boundary between gene expression domains.

<p>(A) Schematic illustration of the model of mutual repression and mobility. (B) The phase plane of the mutual repression system. Blue and red lines indicate nullclines of <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003655#pgen.1003655.e001" target="_blank">equations (1)</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003655#pgen.1003655.e002" target="_blank">(2)</a>, respectively, without the diffusion terms. The filled circles with magenta and green indicate the stable steady states, named the <i>AD</i>-expressing state and the <i>AB</i>-expressing state, respectively. The open circle is an unstable steady state. (C–E) Simulation results of the mathematical model using three parameter sets: the symmetric parameter set between <i>AD</i> and <i>AB</i> (C); the asymmetric parameter sets (D, E). The parameter values are set to be <i>p₁</i> = <i>p₂</i> = 0.1, <i>r₁</i> = <i>r₂</i> = 2.0, <i>d₁</i> = <i>d₂</i> = 0.1, <i>c₁</i> = <i>c₂</i> = 2.0 and <i>D_AD</i> = <i>D_AB</i> = 0.1 (C), except for <i>r₂</i> = 1.8 (D), <i>r₁</i> = 1.8 (E).</p

The boundary between the <i>FIL</i>-expression and miR165/166-free domains shifts during leaf development.

<p>(A–F) Confocal images of longitudinal (A–C) and transverse (D–F) sections showing <i>FILpro:GFP</i> (green) and <i>35Spro:miYFP-W</i> (magenta) marker expression patterns at different stages: 50-µm-long (A), 200-µm-long (B) and 300-µm-long (C–F). Lower schematic illustrations represent each boxed region in (A–C). (D–F), A series of sections from a leaf of approximately 300 µm in height. The approximate heights of the observation plane from the leaf base are indicated in (D–F). Arrowheads indicate the distal (A–C) and marginal (D–F) tip cells. (G–R) Confocal (G, Q, R) and stereoscopic (H–P) images showing VENUS expression patterns (yellow and yellow-green) of the <i>FILpro:CRE-GR 35Spro:loxP-Ter-loxP-VENUS</i> system in the third leaves of 12-day-old plants. The timing of DEX treatment for CRE/loxP recombination is indicated at the bottom left of each panel. The confocal imaging planes are a transverse section of a shoot apex (G) and third leaves (Q, R). The red color represents chlorophyll fluorescence. “+” marks the meristem center in all figures. Scale bars represent 50 µm (A–G), 1 mm (H–P) and 100 µm (Q, R). ad, adaxial side; ab, abaxial side.</p

The <i>enf2</i> mutant shows slow FMB shifting and an abaxialized leaf phenotype.

<p>(A–E) Confocal images of transverse sections showing <i>FILpro:GFP</i> (green) and <i>35Spro:miYFP-W</i> (magenta) marker expression in the wild-type (A) and <i>enf2</i> (B–E) leaf primordia. The arrowheads indicate the provascular cells. (C–E), A series of sections from a leaf of approximately 300 µm in height. The approximate heights of the observation plane from the leaf base are indicated. The comparable WT data are <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003655#pgen-1003655-g002" target="_blank">Figure 2D–F</a>. (F) <i>FIL</i>-expression area sizes (%, y-axis) at different stages (grouped by section area sizes, x-axis) of the wild-type and <i>enf2</i> leaf primordia. Bars indicate standard errors. n.s., not significantly different; *, significantly different (<i>p</i><0.05, t-test) between the wild type and <i>enf2</i>. (G, H) Seedlings of the wild type and <i>enf2</i>. (I, J) Scanning electron microscope images of leaf sections from the wild type and <i>enf2</i>. Scale bars represent 50 µm (A–E, I, J) and 1 mm (G, H). WT, wild type.</p

The plastid effects on FMB shifting and lamina expansion depend on the <i>GUN1</i> gene.

<p>(A–C) Seedlings of lincomycin-treated <i>gun1</i>, untreated <i>enf2 gun1</i> and untreated <i>gun1</i>. (D–F) Confocal images of transverse sections showing <i>FILpro:GFP</i> (green) and <i>35Spro:miYFP-W</i> (magenta) marker expression in leaf primordium of each above plant. The arrowheads indicate the provascular cells. Scale bars represent 1 mm (A–C) and 50 µm (D–F).</p

FMB shifting is quicker in the <i>phb-1d/+</i> mutant than in the wild type.

<p>(A–E) Confocal images of longitudinal (A) and transverse (B–E) sections showing <i>FILpro:GFP</i> (green) and <i>35Spro:miYFP-W</i> (magenta) marker expression in <i>phb-1d/+</i> leaf primordia. (C–E), A series of transverse sections from a leaf of approximately 300 µm in height. The comparable WT data are <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003655#pgen-1003655-g002" target="_blank">Figure 2D–F</a>. (F, G) VENUS expression pattern (yellow and yellow-green) of <i>FILpro:CRE-GR 35Spro:loxP-Ter-loxP-VENUS</i> in <i>phb-1d/+</i>. A transverse section of leaf primordia (F) and a stereoscopic image of mature leaf (G). (H, I) Seedlings of the wild type and <i>phb-1d/+</i>. (J, K) Scanning electron microscope images of leaf sections in the wild type and <i>phb-1d/+</i>. Scale bars represent 50 µm (A–F), 1 mm (G–I) and 100 µm (J, K).</p

Inhibition of plastid gene expression machinery retards FMB shifting and leads to narrow lamina formation.

<p>(A–D) Seedlings of lincomycin- and erythromycin- treated wild type, and untreated <i>flv</i> mutant and wild type. (E–H) Confocal images of transverse sections showing <i>FILpro:GFP</i> (green) and <i>35Spro:miYFP-W</i> (magenta) marker expression in leaf primordium of each above plant. The arrowheads indicate the provascular cells. Scale bars represent 1 mm (A–D) and 50 µm (E–H). Lin, lincomycin; Ert, erythromycin.</p