Robust organ size in Arabidopsis is primarily governed by cell growth rather than cell division patterns

Organ sizes and shapes are highly reproducible, or robust, within a species and individuals. Arabidopsis thaliana sepals, which are the leaf-like organs that enclose flower buds, have consistent size and shape, which indicates robust development. Counterintuitively, variability in cell growth rate over time and between cells facilitates robust development because cumulative cell growth averages to a uniform rate. Here we investigate how sepal morphogenesis is robust to changes in cell division but not robust to changes in cell growth variability. We live image and quantitatively compare the development of sepals with increased or decreased cell division rate (lgo mutant and LGO overexpression, respectively), a mutant with altered cell growth variability (ftsh4), and double mutants combining these. We find that robustness is preserved when cell division rate changes because there is no change in the spatial pattern of growth. Meanwhile when robustness is lost in ftsh4 mutants, cell growth accumulates unevenly, and cells have disorganized growth directions. Thus, we demonstrate in vivo that both cell growth rate and direction average in robust development, preserving robustness despite changes in cell division.

LGOoe (F).The heat map scale is 1 to 15 daughter cells and the scale bar is 50µm.(G-R) Heat maps of number of daughter cells per lineage over 24-hour intervals for remaining two replicates of WT (G-H), lgo-2 (I-J), LGOoe (K-L), ftsh4-5 (M-N), lgo-2 ftsh4-4 (O-P), and LGOoe (Q-R).The lowest heat map value represents 1 cell per lineage, which means no division.The greatest heat map value represents 4 or more cells per lineage.The scale bar is 50µm.Heat maps are projected onto the later time point.Related to Figures 2 and 4.

Figure S3 :
Figure S3: Cell sizes remain smaller in lgo-2, and lgo-2 ftsh4-5, and become progressively larger in LGOoe and LGOoe ftsh4.(A-L) Heat maps of cell area at each image time point for remaining two replicates of WT (A-B), lgo-2 (C-D), LGOoe (E-F), ftsh4-5 (G-H), lgo-2 ftsh4-4 (I-J), and LGOoe (K-L).The heat map scale is 0 to 4200µm 2 and the scale bar is 50µm.(M) Density plots showing how the distribution of cell areas for each genotype changes over time.Related to Figure 3 and SupplementalFigure S4.

Figure S4 :
Figure S4: Cell size diverges over time due to differences in cell division.Multidimensional scaling (also called principal coordinate analysis) is used to represent the distributions of cell areas for each genotype and time interval in two-dimensional space.Increased distance between points indicates larger differences in the cell size distributions whereas points clustered together indicates that these cell size distributions are similar.Colors and shapes represent genotypes and time intervals, respectively.Note the cell size distributions of all genotypes cluster at the 0 hr and 24 hr time points, but then spread out based on the cell division rate.Related to Figure 3 and Supplemental Figure S2.

Figure S5 :
Figure S5: Cell growth follows a basipetal gradient which is preserved when cell division changes, but altered in ftsh4-5, lgo-2 ftsh4-5, and LGOoe ftsh4-5.(A-L) Heat maps of cell area growth over each 24-hour interval that are projected onto the later time point for the remaining two replicates of WT (A-B), lgo-2 (C-D), LGOoe (E-F), ftsh4-5 (G-H), lgo-2 ftsh4-5 (I-J), and LGOoe ftsh4-5 (K-L).The heat map represents the change in ratio of cell area (cell area of later time point divided by cell area of earlier time point) and the scale is 1 to 3. Localization of fast growth is marked by red outlines, and is band-like in WT, lgo-2, and LGOoe and patchy in ftsh4-5, lgo-2 ftsh4-5, and LGOoe ftsh4-5.The scale bar is 50µm.

Figure S6 :
Figure S6: Distributions of cell growth cluster loosely based on developmental time, and not by genotype.Multidimensional scaling (also called principal coordinate analysis) is used to represent the distributions of cell area growth rates for each genotype and time interval in two-dimensional space.Increased distance between points indicates larger differences between the growth rate distributions whereas points clustered together indicates that these distributions are similar.Colors and shapes represent genotypes and time intervals, respectively.Related to Figure 5.

Figure S7 :
Figure S7: Cell lineages that are dividing have slightly faster growth rates.Distribution of cell growth ratio for populations of cells that have divided a specified number of times over a 24-hour interval are plotted for each genotype and time interval.Note that at early time points cell lineages that have undergone more divisions also tend to exhibit higher growth rates, but at later time points the growth curves are more equivalent.Related to Figure 5.

Figure S8 :
Figure S8: Cells can have regions with different growth rates in both WT and LGOoe.(A-D) Giant cells are artificially subdivided into multiple cells and outlined in white.The heat maps of cell area growth over each 24-hour interval are projected onto the later time point for remaining two replicates of WT (A-B) and LGOoe (C-D).The heat map represents the change in ratio of cell area (cell area of later time point divided by cell area of earlier time point) and the scale is 1 to 3. The scale bar is 50µm.Related to Figure 6.