Regulatory circuit modeling two-dimensional patterns of stomatal initial cells.

<p>(<b>A</b>) Diagram outlining the regulatory circuit used for modeling. (Top) Example of two adjacent protodermal cells undergoing fate determination process. Arrow designates activation and T-bar designates inhibition. Concentrations of each components are abbreviated as the following: <i>u</i>₁, SPCH; <i>u</i>₂, SCRM; <i>u</i>₃, SPCH•SCRM heterodimer; <i>v</i>₁, EPF2; <i>w</i>, TMM; <i>v</i>₂, EPF2-independent hypothetical component, most likely BR pathway; <i>m</i>, strength of MAPK cascade-mediated inhibition. <u><i>S</i></u>, a component that competes for receptor pools, most likely Stomagen. The site of bikinin action is also indicated. Initially, all cells possess and operate identical regulatory circuit. Stochastic noise will be amplified in such a way that a cell expressing more activator will self-activate its stomatal-lineage character (light blue), while the neighboring cell will lose stomatal-lineage character (white). The regulatory relationships that are not experimentally verified are in green. It is not known which protodermal cells produce BR, or whether BR acts in neighboring cells. (Bottom) Simplified diagram showing the putative range of inhibitor action. (<b>B</b>) Spatial patterns of each component in wild-type and each mutant background simulated <i>in silico</i> based on the mathematical models. Each square represents a sheet of protoderm with 400 cells (each cell represented by a hexagon). White cells indicate no expression/accumulation of a given component, while dark-blue cells express/accumulate high amounts.</p

Differential regulation of receptors by SPCH•SCRM module.

<p>(<b>A</b>) Expression/accumulation patterns of functional ERECTA-YFP (top) and TMM-YFP (bottom) in protoderm from first rosette leaf primordia of 5-8-day-old <i>erecta tmm</i> (left), <i>spch</i> (middle), and <i>scrm scrm2</i> (right) seedlings. No TMM-YFP signal can be detected in the absence of <i>SPCH</i> or <i>SCRMs</i>. Scale bars, 150 μm. (<b>B</b>) qRT-PCR analysis of <i>EPF2</i>, <i>TMM</i>, <i>ERECTA</i>, and <i>STOMAGEN</i> transcripts levels from five-day-old <i>spch</i> (pavement cells only), <i>scrm scrm2</i> (pavement cells only), <i>scrm-D mute</i> (meristemoid enriched), and <i>scrm-D</i> (stomata enriched) seedlings compared to wild-type. Both <i>EPF2</i> and <i>TMM</i> transcripts are highly enriched in meristemod-enriched population (<i>scrm-D mute</i>) while undetectable in <i>spch</i> or <i>scrm scrm2</i>. In contrast, <i>ERECTA</i> and <i>STOMAGEN</i> show no such trends. (<b>C</b>) Higher magnifications of protoderm expressing ERECTA-YFP levels (top left and middle) and TMM-YFP (top right) co-stained with PI (middle) to highlight cell periphery. Presented at the bottom are line scan analyses of each panel corresponding to lines indicated in the confocal images. Cell boundaries between a stomatal-lineage cell and an adjacent epidermal cell (asterisks), between a meristemoid and an SLGC (x), between a GC and adjacent epidermal cells (v), and between two paired GCs (+) are indicated. ERECTA-YFP levels are reduced in stomatal precursors and not detectable in GCs, while TMM-YFP levels are stomatal-lineage-specific (<b>D</b>) ChIP assays on <i>TMM</i> promoter region using anti-GFP antibody on control Col-0 or transgenic seedlings expressing functional SPCH-GFP in <i>scrm-D</i>, GFP-SCRM, GFP-scrm-D, or GFP-SCRM2. Each amplicon is indicated by a letter. Shown are the means ± SEM of fold enrichment over wild type Col from three biological replicates. Line, intergenic region or intron; arrow, transcription start site; filled rectangle, coding region. (<b>E</b>) Transactivation dual luciferase reporter assays using <i>N</i>. <i>benthamiana</i>. <i>TMM</i> expression is upregulated when both SPCH and SCRM are present. Bars indicate means of triplicate; error bars, S.E.M.</p

Bikinin treatment represses GFP-SCRM accumulation independent of EPF2-and ERECTA-family.

<p>The bikinin-sensitive, EPF2-independent pathway may constitute the second feedback loop predicted by our modeling. (<b>A-D</b>) wild-type seedlings carrying <i>SCRM</i>::<i>GFP-SCRM</i> mock treated (A, B) or treated with 30 μM bikinin (C, D). (<b>E-H</b>) <i>epf2</i> seedlings carrying <i>SCRM</i>::<i>GFP-SCRM</i> mock treated (E, F) or treated with 30 μM bikinin (G, H). (<b>I-L</b>) wild-type seedlings carrying <i>SCRM</i>::<i>GFP-scrm-D</i> mock treated (I, J) or treated with 30 μM bikinin (K, L). (<b>M-P</b>) <i>er erl1 erl2</i> seedling carrying <i>SCRM</i>::<i>GFP-SCRM</i> mock treated (M, N) or treated with 30 μM bikinin (O, P). (<b>Q-T</b>) wild-type seedlings carrying <i>AtML1</i>::<i>nucGFP</i> mock treated (Q, R) or treated with 30 μM bikinin (S, T). Shown are 5-day-old cotyledon epidermis (A, C, E, G, I, K, M, O, Q, S) and protoderm of primary leaf primordial (B, D, F, H, J, L, N, P, R, T) after 2-day exposure to bikinin. Under bikinin treatment, GFP-SCRM signal disappears from stomatal precursors (arrowheads), while GFP-SCRM in stomata (asterisks) is still detected. Reduction of the GFP-SCRM signal was evident ~ 8 hrs after bikinin treatment and the signals became undetectable 2 days after treatment. For cotyledons, cell periphery was highlighted by propidium iodide; scale bars, 50 μm. For primary leaves, scale bars, 100 μm.</p

Molecular framework of the negative-feedback loop between SPCH•SCRM and EPF2 for stomatal-lineage specification.

<p>(<b>A</b>) Shown are confocal images of abaxial protoderm of rosette leaf primordia of 10-11-day-old seedlings expressing <i>EPF2pro</i>::<i>erGFP</i> in wild type (left), <i>spch</i> (middle), and <i>scrm scrm2</i> (right). No <i>EPF2</i> promoter activity is detected in the absence of <i>SPCH</i> or <i>SCRMs</i>. Scale bars, 20 μm. (<b>B</b>) ChIP assays on <i>EPF2</i> promoter region using anti-GFP antibody on control Col or transgenic seedlings expressing functional SPCH-GFP in <i>scrm-D</i>, GFP-SCRM, GFP-scrm-D, or GFP-SCRM2. Each amplicon is indicated in a red letter. Mean ± SEM of fold enrichment over wild-type Col from three biological replicates are shown. <i>ACT2</i> serves a control. Line, intergenic region or intron; arrow, transcription start site; filled rectangle, coding region. (<b>C</b>) Transactivation dual luciferase reporter assays in <i>N</i>. <i>benthamiana</i>. Strong <i>EPF2</i> reporter expression is detected when both SPCH and SCRM are present. Bars indicate means of biological triplicates; error bars, S.E.M. (<b>D</b>) Effects of bioactive recombinant MEPF2 peptide application on promoter activity and protein accumulation of SPCH and SCRMs. MEPF2 application has no effect on <i>SPCH</i> promoter activity (<i>SPCHpro</i>::<i>nucGFP</i>) despite the fact that no-stomatal cell linages are initiated (top left). In contrast, MEPF2 application results in loss of GFP signals in <i>SPCHpro</i>::<i>SPCH-GFP</i> (top right), <i>SCRMpro</i>::<i>nucGFP</i> (middle left), <i>SCRMpro</i>::<i>GFP-SCRM</i> (middle right), and <i>SCRM2pro</i>::<i>GFP-SCRM2</i> (bottom left). GFP-scrm-D protein is insensitive to MEPF2 application (bottom right). Six-day-old cotyledons are imaged under the same magnification. Scale bar, 20 μm. (<b>E</b>) Abaxial epidermis from 5-6-day-old seedling rosette leaf primordia expressing <i>SPCHpro</i>::<i>SPCH-GFP</i> in wild-type (left) or <i>scrm-D</i> (right) background, showing that more protodermal cells accumulate SPCH-GFP protein (green) in <i>scrm-D</i>. Scale bar, 20 μm.</p