Fluctuations of the transcription factor ATML1 generate the pattern of giant cells in the Arabidopsis sepal
- Cornell University, United States
- University of Cambridge, United Kingdom
- Univ Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRA, France
- Lund University, Sweden
- Microsoft Research, United Kingdom
Figures
Figure 1
The scattered pattern of giant epidermal cells.
(A) An image of a wild-type (WT) Arabidopsis thaliana flower. The sepals (s) are the outermost leaf-like floral organs. (B) SEM image of developing sepals on young flower buds. The three flowers in …
Figure 2 with 1 supplement
ATML1 levels influence the quantity of giant cells that form on the sepal.
(A–F) SEM images of sepals from an ATML1 genetic dosage series. Giant cells are false colored in red. (A) ATML1 overexpression line that is homozygous for the pPDF1::FLAG-ATML transgene. (B) ATML1 …
Figure 2—figure supplement 1
ATML1 estradiol inducible transgenic plants form ectopic giant cells five days after application of 10 µM estradiol.
(A) A confocal image of an untreated ATML1 estradiol-inducible stage 10 flower expressing an ATML1 transcriptional marker (proATML1-nls-3XGFP). Note that ATML1 transcriptional reporter is only …
Figure 3 with 1 supplement
mCitrine-ATML1 expression is variable from cell to cell in the sepal but uniform in the meristem.
(A) SEM image of a wild-type (Col) sepal. (B) SEM image of an atml1–3 mutant sepal. Note that atml1 mutants exhibit a lack-of-giant-cell phenotype. (C–D) SEM images showing that the pATML1::mCitrine-…
Figure 3—figure supplement 1
The transcriptional reporter SEC24A:: H2B-GFP and the fusion proteins VIP1-mCitrine, and AP2-2XYpet are uniformly expressed in the developing sepal.
(A) Confocal denoised images of three developing sepals expressing pSEC24A::H2B-GFP. (B) Heat maps of normalized mean concentration levels of pSEC24A::H2B-GFP expression in the developing flowers. (C…
Box 1—Figure 1
Image analysis pipeline to quantify fluorescent fusion protein concentration.
(A) Raw confocal image of developing sepal expressing mCitrine-ATML1 (sepal also presented in Figure 4). (B) Denoised confocal images using PureDenoise ImageJ software. (C) Binary mask created in …
Figure 4 with 5 supplements
ATML1 fluctuates in sepal epidermal cells to initiate giant cell patterning.
(A) Raw images of pATML1::mCitrine-ATML1 (white) from a live imaging series of a developing sepal. Images were taken every 8 hr for 64 hr. (B) Heat map showing corresponding mCitrine-ATML1 …
Figure 4—figure supplement 1
Second flower that demonstrates ATML1 fluctuates in sepal epidermal cells to initiate giant cell patterning.
(A) Raw images of pATML1::mCitrine-ATML1 (white) from a live imaging series of a developing sepal. Images were taken every 8 hr for 40 hr. (B) Heat maps showing corresponding mCitrine-ATML1 …
Figure 4—figure supplement 2
Third flower that demonstrates ATML1 fluctuates in sepal epidermal cells to initiate giant cell patterning.
(A) Raw images of pATML1::mCitrine-ATML1 (white) from a live imaging series of a developing sepal. Images were taken every 8 hr for 64 hr. (B) Heat map showing corresponding mCitrine-ATML1 …
Figure 4—figure supplement 3
Fourth flower that demonstrates ATML1 fluctuates in sepal epidermal cells to initiate giant cell patterning.
(A) Raw images of pATML1::mCitrine-ATML1 (white) from a live imaging series of a developing sepal. Images were taken every 8 hr for 64 hr. (B) Heat map showing corresponding mCitrine-ATML1 …
Figure 4—figure supplement 4
Giant cells can be identified by their large, elongated, endoreduplicating nuclei.
(A) Confocal image of two sepals expressing pATML1::mCitrine-ATML1 (Green) in the nucleus and the plasma membrane marker pML1:mCherry-RCI2A (Red). Asterisks mark giant endoreduplicating cells. Note …
Figure 4—figure supplement 5
Mean normalized mCitrine-ATML1 concentrations for all four pATML1::mCitrine-ATML1;atml1–3 flowers.
(A) mCitrine-ATML1 flower number 1 (shown in Figure 4). Flower has an inferred normalized ATML1 concentration peak threshold of 1.21. (B) mCitrine flower number 2 (shown in Figure 4—figure …
Box 2—Figure 1
Nuclear area was used to determine cell cycle stage.
(A) DAPI stained wild-type sepal nuclei show that DNA content and nuclear area are linearly correlated (R2 = 0.903). 2C nuclei are colored yellow, 4C nuclei are colored blue, and 8C/16C nuclei are …
Figure 5 with 2 supplements
A threshold-based mechanism is consistent with increased giant cell formation in ATML1 overexpression lines.
(A) Raw images of pPDF1::GFP-ATML1 (white) from a live imaging series of a developing overexpression sepal. Images were taken every 8 hr for 48 hr. (B) Heat map showing corresponding GFP-ATML1 …
Figure 5—figure supplement 1
Second flower demonstrating that a threshold-based mechanism is consistent with increased giant cell formation in ATML1 overexpression lines.
(A) Raw images of pPDF1::GFP-ATML1 (white) from a live imaging series of a developing overexpression sepal. Images were taken every 8 hr for 56 hr. (B) Heat maps showing corresponding GFP-ATML1 …
Figure 5—figure supplement 2
Third flower demonstrating that a threshold-based mechanism is consistent with increased giant cell formation in ATML1 overexpression lines.
(A) Raw images of pPDF1::GFP-ATML1 (white) from a live imaging series of a developing overexpression sepal. Images were taken every 8 hr for 56 hr. (B) Heat maps showing corresponding GFP-ATML1 …
Figure 6 with 2 supplements
The dynamics of ATML1 fluctuations are independent of endoreduplication.
(A) Raw images of pATML1::mCitrine-ATML1 (white) from a live imaging series of a developing lgo mutant sepal. Images were taken every 8 hr for 64 hr. (B) Heat maps showing corresponding …
Figure 6—figure supplement 1
Second flower showing that dynamic fluctuations of ATML1 are independent of endoreduplication.
(A) Raw images of pATML1::mCitrine-ATML1 (white) from a live imaging series of a developing lgo mutant sepal. Images were taken every 8 hr for 64 hr. (B) Heat map showing corresponding …
Figure 6—figure supplement 2
Third flower showing that dynamic fluctuations of ATML1 are independent of endoreduplication.
(A) Raw images of pATML1::mCitrine-ATML1 (white) from a live imaging series of a developing lgo mutant sepal. Images were taken every 8 hr for 64 hr. Labels below the snapshots display the time …
Figure 7 with 4 supplements
A plausible stochastic model for giant cell patterning.
(A) Schematic diagram of the computational model for giant cell patterning. Top panel shows the proposed ATML1 model network in which ATML1 can prevent cell division and instead drive entry into …
Figure 7—figure supplement 1
Simulation results showing different stochastic time courses.
Time courses for cells committing to the (A and C) giant fate and (B and D) small cell fate of ATML1 (left), its target (middle left for A and B; middle for C and D) and the timer (middle right for A…
Figure 7—figure supplement 2
Stochastic fluctuations are essential for generating the giant cell patterning.
Phase diagrams across the parameter space of basal ATML1 production rates and ATML1 auto-induction rates showing (A and C) the fraction of giant cells in the tissue and (B and D) the CVs of the …
Figure 7—figure supplement 3
Classification analysis of the simulated data shows that a weak feedback or no feedback in ATML1 reproduces the experimental observations.
Analysis for (A–D) full and (F–J) coarse grained simulated time courses show we get equivalent AUC values and similar ATML1 soft thresholds. (A and F) AUC values of 5 simulations with different …
Figure 7—figure supplement 4
Theoretical and experimental study of the ATML1 auto-induction strength.
(A–D) Simulation results of the model with different ATML1 auto-induction strengths show different qualitative behaviors. Simulations with different feedback strengths and different ATML1 basal …
Figure 8
The model recapitulates ATML1 dosage dependency.
(A) Snapshots showing the resulting patterns of giant cells (8C, 16C, 32C and 64C cells) and small cells (2C and 4C cells) at the final time point of the simulations when the basal ATML1 production …
Figure 9 with 1 supplement
Fluctuations of ATML1 around a soft threshold pattern giant cells and small cells in the sepal.
ATML1 fluctuates in every young sepal epidermal cell. However, cells only respond to high levels of ATML1 during G2 phase of the cell cycle. (A) Schematic showing that in G1, cells are impervious to …
Figure 9—figure supplement 1
ACR4 and DEK1 act in the giant cell patterning pathway.
(A–B) SEM images of a sepal overexpressing (OX) ATML1 under the PDF1 promoter (pPDF1::FLAG-ATML1). (C–D) SEM images of a wild-type sepal. (E–F) SEM images of sepal homozygous for both ATML1 OX …
Videos
Video 1
A movie of a developing pATML1::mCitrine-ATML1; atm1l-3 sepal shown in Figure 4.
The sepal primordium was live imaged every 8 hr until giant cells form.
Video 2
A movie of a developing pATML1::mCitrine-ATML1; atm1l-3 sepal shown in Figure 4—figure supplement 1.
The sepal primordium was live imaged every 8 hr until giant cells form.
Video 3
A movie of a developing pATML1::mCitrine-ATML1; atm1l-3 sepal shown in Figure 4—figure supplement 2.
The sepal primordium was live imaged every 8 hr until giant cells form.
Video 4
A movie of a developing pATML1::mCitrine-ATML1; atm1l-3 sepal shown in Figure 4—figure supplement 3.
The sepal primordium was live imaged every 8 hr until giant cells form.
Box 2—Video 1
A movie of a developing pATML1::mCitrine-ATML1; atml1–3 sepal.
The sepal primordium was live imaged every hour to capture the size (area) of nuclei before and after division. Associated with Box 2.
Box 2—Video 2
A movie of a developing pATML1::mCitrine-ATML1; atml1–3 sepal.
The sepal primordium was live imaged every hour to capture the size (area) of nuclei before and after division. Associated with Box 2.
Video 5
A movie of a developing pPDF1::GFP-ATML1 sepal shown in Figure 5.
The sepal primordium was live imaged every 8 hr until giant cells form.
Video 6
A movie of a developing pPDF1::GFP-ATML1 sepal shown in Figure 5—figure supplement 1.
The sepal primordium was live imaged every 8 hr until giant cells form.
Video 7
A movie of a developing pPDF1::GFP-ATML1 sepal shown in Figure 5—figure supplement 2.
The sepal primordium was live imaged every 8 hr until giant cells form.
Video 8
A movie of a developing pATML1::mCitrine-ATML1; lgo sepal shown in Figure 6.
The sepal primordium was live imaged every 8 hr throughout development.
Video 9
A movie of a developing pATML1::mCitrine-ATML1; lgo sepal shown in Figure 6—figure supplement 1.
The sepal primordium was live imaged every 8 hr throughout development.
Video 10
A movie of a developing pATML1::mCitrine-ATML1; lgo sepal shown in Figure 6—figure supplement 2.
The sepal primordium was live imaged every 8 hr throughout development.
Video 11
Simulation results showing ATML1, target, timer levels and cell ploidies throughout time in a growing tissue.
Cells that cannot divide, increase their ploidy, becoming giant cells. The time resolution of the displayed movie (0.5) is lower than the actual simulation time step (0.1), so fluctuations in ATML1 …
Tables
Table 1
Main parameter values used for simulations in Figures 7 and 8 and Figure 7—supplements 1–4. We omit time and concentration units, since all are considered arbitrary.
Parameter | Description | Values |
|---|---|---|
PA | ATML1 basal production rate | 1.14 |
VA | ATML1 auto-induction rate | 1.25 |
KA | ATML1 concentration for half ATML1 auto-induction maximal rate | 1.9 |
nA | Hill coefficient for ATML1 auto-induction | 5 |
GA | ATML1 degradation rate | 1 |
VT | Target maximal production rate | 10 |
KT | ATML1 concentration for half ATML1-mediated target maximal production rate | 2 |
nT | Hill coefficient for ATML1-mediated target induction | 1 |
GT | Target degradation rate | 10 |
ΘT | Target threshold for inhibiting mitosis | 0.6 |
ΘC,S | Timer threshold for synthesis | 2 |
ΘC,D | Timer threshold for timer resetting | 3 |
PC | Timer basal production rate | 0.1 |
E0 | Characteristic effective volume | 15 |
Exponential radial growth rate | 0.007 | |
Exponential added growth rate to the vertical direction | 0.012 |
Additional files
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Supplementary file 1
A zip file containing both Raw data and selected lineages for pATML1::mCitrine-ATML1 (mCitrine-ATML1), PDF1::GFP-ATML1 (PDF1), and lgo-2;pATML1::mCitrine-ATML1 (lgo) flowers.
See readme files within the different folders for further information. All raw image confocal tif files and example image processing files may be downloaded from: http://dx.doi.org/10.7946/P29G6M
- https://doi.org/10.7554/eLife.19131.046
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Source code 1
MATLAB code for all image quantification and analysis, as well as receiver operator characteristics (ROC) analysis as described in the Materials and methods section.
- https://doi.org/10.7554/eLife.19131.047
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Source code 2
Code for simulating ATML1 dynamics in a growing tissue.
Scripts for the analysis and representation of the simulation results are also provided. See readme files within the different folders for further information.
- https://doi.org/10.7554/eLife.19131.048
