Basic layout of the shoot apical meristem and our model.

<p>A) Schematic representation of the SAM. The central zone (CZ) is located in the center of the shoot apex and contains the stem cells (SCs). <i>CLV3</i> expression marks the SC domain. Directly below the SC domain is the organizing center (OC), which is defined by the expression of <i>WUS</i>. Lateral to the CZ is the peripheral zone (PZ), which consists of rapidly proliferating cells. <i>STM</i> expression in the three outer cell layers partially correlates with the PZ. The surface width of the <i>STM</i> expression domain (dashed line) as well as the surface distance between opposing primordia (solid line) is indicated. B) Schematic representation of the stem cell pool model. Cells of the SC pool (<i>S</i>) and the OC (<i>O</i>) proliferate with rate α₁. SCs differentiate into cells of the OC with rate <i>λ</i>₁ and into cells of the proliferation zone P with rate <i>λ</i>₂. Cells of the proliferation zone can re-specify into SCs with a rate <i>ρ</i>₁. This rate depends on signals from the OC as indicated by the dotted line. Cells of the OC terminally differentiate with a rate <i>λ</i>₃, which depends on the SC pool.</p

Expression of marker genes.

<p>Representative slides of <i>in situ</i> hybridizations using <i>CLV3</i>, <i>WUS</i>, <i>STM</i> and <i>HISTONE H4</i> RNA probes on tissue grown under three different environmental conditions. First row: vegetative meristems form short days, 23°C. Second row: transition meristems from long days, 16°C. Third row: inflorescence meristems from long days, 23°C.</p

Mean and standard deviation of the re-specification rate <i>ρ</i>₁ and the cell proliferation rate of the CZ (<i>α</i>₁) and of the PZ (<i>α</i>₂) in the three investigated conditions.

<p>Parameter <i>ρ</i>₁ was estimated from the data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003553#pone.0003553-Reddy1" target="_blank">[8]</a> and <i>α</i>₁ and <i>α</i>₂ were estimated from our <i>STM in situ</i> data as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003553#s4" target="_blank">Methods</a>. Standard deviations were calculated by error propagation.</p

Quantification of SAM markers.

<p>Box-plot representation of data derived by image analysis from SAM measurements and <i>in situ</i> hybridizations with <i>CLV3</i>, <i>WUS</i>, <i>STM</i> and <i>HISTONE H4</i> RNA probes. A) Area of the <i>CLV3</i> and <i>WUS</i> expression domain. B) Width of the <i>STM</i> expression domain and distance between opposing primordia both measured along the apex surface (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003553#pone-0003553-g001" target="_blank">Figure 1A</a>). C) Mitotic index (MI) of the CZ and the PZ. Mean and standard deviation of the data is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003553#pone-0003553-t001" target="_blank">Table 1</a>. VM: vegetative meristem from short days, 23°C; TM: transition meristem from long days, 16°C; IM: inflorescence meristem from long days, 23°.</p

Cell pool sizes in response to changes in <i>CLV3</i> or <i>WUS</i> activity.

<p>The responses to elevated <i>CLV3</i> signaling are indicated by solid lines, the effect of ectopic <i>WUS</i> expression are denoted by dashed lines. All domain sizes are given relative to the WT pool sizes denoted by <i>S</i>₀ (SC) and <i>O</i>₀ (OC), respectively. Note that only the basic model Equations (5–6) can recover the experimental phenotypes. Here, enhanced <i>CLV3</i> signaling, simulated by increasing <i>λ</i>₃, leads to a reduced OC and SC pool size. Ectopic <i>WUS</i> over-expression, simulated by including a constant SC production rate, enlarges the SC domain and leads to a decrease of the endogenous <i>WUS</i> activity. Black: basic model Equations (5–6). Red: SC-based feedback model Equations (9–10). Blue: OC-based feedback model Equations (9–10).</p

Dependence of the CZ cell output rate and SC pool size.

<p>The output rate is defined by the fraction of SCs that differentiate into PZ cells per unit time. Note that while the basic model Equations (1–2) show a linear increase in SZ size with, both models including a feedback on the SZ differentiation rate exhibit a reduced (Equations (5–6)) or almost absent (Equations (9–10)) change in the SC pool size while delivering the same increase in cell output rate. Circles: vegetative meristem from short days, 23°C. Diamonds: transition meristem from long days, 16°C. Squares: Inflorescence meristem from long days, 23°C.</p

Median and 95% confidence interval for differentiation rates of the three alternative models.

<p>Median and confidence intervals were determined by bootstrapping of the data (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003553#s4" target="_blank">Methods</a>). The <i>χ</i>² value of the basic model Equations (1–2) is 3.9651, of the SC-based feedback model Equations (5–6) is 2.6574, and of the OC-based feedback model Equations (9–10) is 1.6162.</p

Mean and standard deviation of the expression area of <i>CLV3</i> and <i>WUS</i> and the width of the <i>STM</i> expression domain.

<p>P-P distance is measured between opposing primordia along the outer SAM surface. The mitotic index is determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003553#s4" target="_blank">Methods</a>. Sample numbers are given in brackets.</p

Phase-plane diagrams of alternative models for the SC and OC pool sizes.

<p>A) Basic model Equations (1–2). B) SC-based feedback model Equations (5–6), C) OC-based feedback model Equations (9–10). All graphs show the and null clines for two different values of the cell proliferation rate <i>α</i>₁. Solid curves: <i>α</i>₁ = 0.0042, as in CZ of vegetative meristems (VM). Dashed curves: <i>α</i>₁ = 0.0217, as in the CZ of transition meristems (TM). The intersection of each pair of null clines corresponds with a steady state (<i>S^˙</i>,<i>O^˙</i>) and is indicated by a black circle and the corresponding meristem type. Note that the increase in the steady state size of the SC pool (<i>S^*</i>) due to an increase in <i>α</i>₁ is smaller in both feedback models (B–C) compared to the basic model in A).</p

Phenotypic Description of Mutants and Transgenic Lines and Localization of TTG–YFP Fusion Protein

<div><p>(A–D) Seed coat mucilage stained with ruthenium red. This staining visualizes the mucilage coat as a diffusely stained zone around the seed indicated by a curly bracket. (A) wild-type ecotype RLD. (B) <i>ttg1–13</i>, no mucilage is seen. (C) <i>ttg1–13</i> pTTG1:TTG1. (D) <i>ttg1–13</i> pTTG1:TTG-YFP.</p> <p>(E) Seed coat color. Upper row: <i>ttg1–13</i> pTTG1:TTG1, <i>ttg1–13</i> pTTG1:TTG1-YFP line #4, <i>ttg1–13</i> pTTG1:TTG1–YFP line #1. Lower row from left to right: wild-type ecotype RLD, strong allele <i>ttg1–13</i>, weak allele <i>ttg1–9</i> and weak allele <i>ttg1–10</i>.</p> <p>(F) pTTG1:TTG1–YFP fluorescence. Strong fluorescence is found in the nucleus, and moderate fluorescence in the cytoplasm. This is particularly good to see in regions containing undifferentiated cells.</p> <p>(G) Western blot analysis to test the integrity of the TTG1–YFP and GFP–NLS fusion proteins. The TTG1–YFP fusion protein (70.5 kDa) is detected as a single band at the expected size (upper arrowhead). This band is not seen in the control lane ttg1–13, and no degradation products were found. Also the GFP–NLS fusion (31 kDa) is detected at the expected size (lower arrowhead).</p></div