Figure 1.
The cell size pattern in the Arabidopsis sepal epidermis.
(A) Wild type Arabidopsis flower with sepals (s). (B, C) Scanning electron micrographs (SEMs) of a mature wild type sepal. Giant cells (false colored red) are interspersed between smaller cells. (D, E) Flow cytometric analysis of the DNA contents of nuclei in the mature wild type sepal epidermis (D, both front and back) and internal cell layers (E) used to derive the probability parameters for the model. Histograms show DNA content of each nucleus. (F) Graph of DNA content (integrated density of DAPI fluorescence) versus cell area (µm2) of mature sepal cells. The trend line of the data is displayed and R2 = 0.82 (n = 47 pavement cells, normalized with fifty-nine guard cells which are known to be 2C). Ploidy of cells is indicated by color (red, 16C; magenta, 8C; green, 4C; and blue, 2C). (G) Conceptual model proposing that the probabilistic entry of cells into endoreduplication at different times generates the cell size pattern. Scale bars: 100 µm.
Figure 2.
Cell size correlates with timing of endoreduplication.
(A, B) Live imaging of the development of a wild type sepal primordium imaged every 6 h for 72 h corresponding to Video S1. Images show epidermal nuclei (pATML1::H2B-mYFP) in gold and cell walls (propidium iodide [PI]) in green. A cell and all of its progeny receive the same colored dot. The sepal is outlined in white, giant cells that fail to divide throughout the sequence are indicated with white arrows, and differentiated guard cells (gc) are indicated with a white asterisk. An example of neighboring giant cell and small cell clones are outlined in yellow (shown in detail in C). Note that these clones grow to the same extent. However, comparing the giant cell outlined in yellow to the giant cell outlined in blue shows that growth throughout the sepal is not equivalent. (C) Tracking the development of the neighboring red giant cell and the brown small cell lineage (outlined in yellow in A–B). Images are at the same magnification. The daughter nuclei resulting from a division are circled in white. Although the red and brown cells are equivalent in size and appearance in the young sepal primordium, the red cell never divides throughout the 72-h sequence and becomes a giant cell. By 48 h the red cell nucleus has started to enlarge, indicating that the cell is endoreduplicating. In contrast, the brown cell progeny have undergone two, three, or four divisions. (D) Graph showing that the area of cells depends on the division pattern. The areas of six progenitor cells (outlined with plasma membrane marker pATML1::mCitrine-RCI2A) from Video S3 (labeled with equivalent colors) were tracked over time showing that cells generally increase in size except when they divide. Note the daughter cell sizes are often not exactly equal and that the cell cycle times are nonuniform. For more details on small cell lineages, see Figure S1. Scale bars: 20 µm. See also Videos S1, S2, S3.
Figure 3.
The Intercalary Growth Model reproduces the cell size distribution.
(A, B) Live imaging of the initiation of a wild type sepal primordium showing that based on lineage analysis the outer sepal epidermis is derived from approximately two rows of 8 cells (outlined in white) (Video S5). Epidermal nuclei (pATML1::H2B-mYFP) are in gold and cell walls (PI) are green. Each cell and all of its progeny are labeled with the same colored dot. A bright dot sitting on the top of the sepal in (B) is a pollen grain. Scale bars: 10 µm. (C) Single frame from live imaging of a lateral sepal (outlined in white) (Video S6). Plasma membranes are also marked in gold (pATML1::mCitrine-RCI2A). Note the top of the sepal contains differentiated guard cells (asterisk gc) and the cells are no longer dividing, whereas cells in the bottom of the sepal are actively dividing (daughters of divisions occurring within the last 6 h are circled in white). Scale bar: 50 µm. (D) Scatter plot showing the vertical position of each division (Video S6) event as a percentage of the sepal length (1 = top and 0 = bottom). Red arrow indicates the progressive basipetal termination of divisions. (E–F) Live imaging of an older sepal primordium imaged every 12 h (E: day 0) through maturity at stage 12 (F: 7.6 d). Cells from the middle of the sepal primordium and their progeny have been tracked throughout the 7-d sequence and have been used to delineate regions of the primordium and corresponding regions of the mature sepal (outlined in white). These regions are arbitrary and depend only on the cells that were chosen for tracking. Note that the top half of the primordium makes only the tip of the mature sepal, whereas the middle of the primordium makes the top half of the sepal, and the bottom few cell layers make the whole bottom half of the mature sepal, indicating that these bottom cells have continued to proliferate. Scale bars: 50 µm. (G) Intercalary Growth (IG) Model. The computational sepal develops from an oversimplified generative layer of 8 cells. In the model, the generative layer cells proliferate throughout development. The upper progeny of the generative layer enter the patterning divisions and terminate after undergoing three cell cycles whether mitotic or endocycles. The final image to the right is at a reduced magnification. Cell color corresponds to ploidy: white, 2C generative layer; blue, 2C; green, 4C; magenta, 8C; and red, 16C. (H) Histogram showing that cells are produced in four exact sizes by the IG model when only endoreduplication is allowed to vary. Cell cycle lengths are constant and divisions are exactly symmetric (compare with panel K). The area axis is scaled at log base 2. (I) Histogram of cell cycle times (in 6-h increments) measured from the wild type live imaging data (Videos S1, S2, S3). (J) Histogram of cell areas in the mature sepal epidermis determined by semi-automated image processing (grey; see Text S1 for details). The area axis is scaled at log base 2. The ploidy of cells is calibrated with the 47 pavement cells for which both DNA content and area are known from Figure 1F (the extent of the region is underlined). Note that cells areas fall in a broad distribution although peaks for 2C and 4C are visible. (K) Histogram of cell areas produced by the IG Model, including variability in the cell cycle time and noise in the symmetry of division, showing that each ploidy level has a distribution of cell sizes. The overall size distribution is not significantly different from the in vivo distribution (Figure 3J) (see Text S1 for further analysis). See also Videos S4–S6.
Figure 4.
The model predicts the phenotypes of plants with altered cell size distributions due to gain or loss of cell cycle inhibitor function.
(A) Increasing the probability of entering endoreduplication (p1 = 0.5) in the first cell cycle of the IG model creates sepals with additional giant cells similar to the phenotype of pATML1::KRP1 sepals. Compare to Figure 3G. Cell color corresponds to ploidy: white, 2C generative layer; blue, 2C; green, 4C; magenta, 8C; and red, 16C. (B) pATML1::KRP1 flower. Note the abnormal outward curvature of the sepals. (C, D) SEMs of a pATML1::KRP1 sepal showing giant cells interrupted by islands of small cells. Giant cells are false colored red. (E) Setting the probability of entering endoreduplication in the first cell cycle to zero (p1 = 0) in the IG model creates sepals without giant cells similar to lgo sepals. Compare with Figure 3G. (F) lgo-1 mutant flower. (G, H) SEMs of a lgo-1 sepal showing the absence of giant cells. (I) Flow cytometry analysis of the epidermal DNA content in wild type, lgo-1, and pATML1::KRP1 sepals. The inset shows an enlargement of 16C graph. Graph shows mean percentages and error bars represent the 95% confidence interval. (J) LGO encodes the cell cycle inhibitor SIAMESE RELATED 1 (SMR1) (AT3g10525). The lgo-1 allele contains a mutation of C to T at base 184, which causes substitution of serine (S) for proline (P) at amino acid 62. The lgo-2 allele contains a T-DNA insertion. Scale bars: 100 µm. See also Videos S7, S8 and Figure S2.
Figure 5.
Cell cycle inhibitors promote early entry into endoreduplication.
Epidermal nuclei (pATML1::H2B-mYFP) are in gold and cell walls (PI) are green. Each cell and all of its progeny are labeled with the same colored dot. Cells that do not divide throughout the image sequence are marked with white arrows and guard cell pairs (gc) are noted with white asterisk. Sepals are outlined in white. Compare with Figure 2. (A–C) Live imaging of a pATML1::KRP1 sepal for 60 h (Video S9). (C) Two endoreduplicating cells grow throughout the time series. (D) Graph comparing the number of rounds divisions undergone by wild type, pATML1::KRP1, and lgo-1 cells during the imaging sequences. Cells that have undergone four or five rounds of division are generally in the stomatal development pathway. (E–G) Live imaging of lgo-1 for 72 h (Video S12). (G) All the cells continue dividing through 48-h time point, whereas in wild type the giant cells have already stopped dividing at the time point equivalent to 12 h. Scale bars: 10 µm. See also Videos S9–S14.
Figure 6.
Changing the cell cycle duration shifts the resultant cell areas.
(A) Comparison of the in vivo wild type (blue; reproduced from Figure 3J) cell size distribution to lgo-1 (red) and pATML1::KRP1 (green). Cell sizes were measured from images with semi-automated segmentation. The giant cells (black arrow) are lacking in lgo-1 and increased in pATML1::KRP1. The overall size distribution is shifted toward larger cell sizes in pATML1::KRP1 and slightly smaller cells in lgo-1. (B) In silico cell size distributions created by adjusting the endoreduplication probability and the cell cycle time distributions in the IG model to replicate lgo-1 and pATML1::KRP1. Note the differences in the large cell peaks and the shift of the overall cell size curves are similar to the in vivo data. (C–D) In vivo histogram of the duration of the cell cycle in lgo-1 (C) and pATML1::KRP1 (D) sepal cells (dark blue bars) fit to a probability distribution (blue curve), showing a trend toward shorter (C) or longer (D) cell cycle times than wild type (red curve reproduced from Figure 3I). Note that in both cases the cell cycle times have 6-h resolution due to the time points in the live imaging.
Figure 7.
Endoreduplication does not increase overall growth of the organ.
(A) Image of wild type, lgo-1, and pATML1::KRP1 plants showing that they grow to approximately the same size. (B) Graph of the average areas of mature whole sepals showing the mutants have small effects on sepal area. Error bars represent the 95% confidence intervals. Scale bar: 1 cm.
Figure 8.
The timing of cell division creates the cell size pattern in the sepals.
(A–C) Hypothetical graphs of the change in cell size over time in wild type (A), overexpression of the cell cycle inhibitor KRP1 (pATML1::KRP1) (B), and loss of cell cycle inhibitor activity in the lgo mutant (C). The size of 2C cells oscillates as they grow and divide. As cells decide to endoreduplicate, they exit the oscillating path and continue to grow throughout the remaining cell cycles. Their ultimate size depends on the cell cycle in which they started to endoreduplicate. Line weight roughly indicates the relative cell numbers. (D) Repeating the patterning cell cycles produces the cell size distribution of the sepal epidermis. The timing of endoreduplication creates the means for each ploidy as indicated by the lines. The range in cell sizes for each ploidy is created by unequal divisions (E) and asynchronous cell cycles (F). (E) Small differences in the sizes of daughter cells after a division add to variability in cell size. (F) The duration of cell cycles is variable. Throughout the cell cycle, the cells continue to grow, such that longer cycles result in larger cells, which add to the variability of cell sizes around the mean established by endoreduplication.