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

Abstract

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.

Keywords: Arabidopsis thaliana; FTSH4; LGO; endoreduplication; giant cells; morphogenesis; sepal; spatiotemporal averaging.

Figure 1:. Organ size and shape is robust to changes in LGO expression, but not in ftsh4–5 and double mutants.

(A-F) Stage 15 mature flowers and (G-L) stage 12 flower buds of WT (A, G), lgo-2 mutant (B, H), LGOoe (C, I; pML1::LGO), ftsh4–5 mutant (D, J), lgo-2 ftsh4–5 double mutant (E, K), and LGOoe ftsh4–5 (F, L). Scale bars are 1 mm. Red arrows point to abnormally shaped sepals. (M) Bar graphs of standard deviation of sepal area within one flower. n=104 (WT), 100 (lgo-2), 104 (LGOoe), 108 (ftsh4–5), 116 (lgo-2 ftsh4–5), 108 (LGOoe ftsh4) sepals. Error bars show the standard error of the mean. Letters mark the groups that are not significantly different. (N-S) Contours of mature outer (abaxial) sepals normalized by size (red lines) and the average sepal shape (black line). n=18 (WT), 23 (lgo-2), 22 (LGOoe), 24 (ftsh4–5), 27 (lgo-2 ftsh4–5), 22 (LGOoe ftsh4) sepals. (T) Boxplots of variability of abaxial sepal shape (S_2 as described in Hong et al 2016). n=77 (WT), 66 (lgo-2), 78 (LGOoe), 85 (ftsh4–5), 96 (lgo-2 ftsh4–5), 74 (LGOoe ftsh4) The boxes extend from the lower to upper quartile values of the data with the midline indicating the median and the whiskers extend past 1.5 × interquartile range. Small dots for each box indicate outliers. Letters mark the groups that are not significantly different. Contours for inner (adaxial) and lateral sepals are available in Supplemental Figure S1.

Figure 2:. Cell division rate is decreased by LGO overexpression (LGOoe) and LGOoe ftsh4 and is increased in lgo-2 and lgo-2 ftsh4–5.

(A-F) Heat maps of number of daughter cells per lineage using lineage tracking from 0-hour time point to 120-hour time point that are projected onto the 120-hour time point for WT (A), lgo-2 (B), LGOoe (C), ftsh4–5 (D), lgo-2 ftsh4–4 (E), and LGOoe (F). The heat map scale is 1 to 15 daughter cells, where 1 indicates that no divisions have taken place because one cell gave rise to one cell at the final time point. The scale bar is 50μm. Representative images from n=3 biological replicates. Additional replicates are available in Supplemental Figure S2A–F. (G) Average number of daughter cells per lineage over the 120hr time lapse imaging. n=3. Error bars are the standard error of the mean. Letters mark the groups that are not significantly different. (H) Average number of cells that do not divide over the 120hr time lapse imaging. n=3. Error bars are the standard error of the mean.

Figure 3:. Cell sizes remain smaller in lgo-2, and lgo-2 ftsh4–5, and become progressively larger in LGOoe and LGOoe ftsh4.

(A-G) Heat maps of cell area at each image time point for WT (A), lgo-2 (B), LGOoe (C), ftsh4–5 (D), lgo-2 ftsh4–5 (E), and LGOoe ftsh4–5 (F). The heat map scale is 0 to 4000μm² and the scale bar is 50μm. Representative images from n=3 biological replicates. Additional replicates are available in Supplemental Figure S3. (D) Distribution of cell areas at each time point. Statistical analysis (multidimensional scaling) available in Supplemental Figure S4.

Figure 4:. Cell division follows a basipetal gradient in WT, lgo-2 and LGOoe, but not in ftsh4–5, lgo-2 ftsh4–5, and LGOoe ftsh4–5.

(A-F) Heat maps of number of daughter cells per cell lineage over 24-hour intervals for WT (A), lgo-2 (B), LGOoe (C), ftsh4–5 (D), lgo-2 ftsh4–5 (E), and LGOoe ftsh4–5 (F). 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. Representative images from n=3 biological replicates. Additional replicates are available in Supplemental Figure S2.

Figure 5:. 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-F) Heat maps of cell area growth over each 24-hour interval that are projected onto the later time point for WT (A), lgo-2 (B), LGOoe (C), ftsh4–5 (D), lgo-2 ftsh4–5 (E), and LGOoe ftsh4–5 (F). 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. Daughter cells that result from a division over a given time interval area outlined in white. 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. Representative images from n=3 biological replicates. Additional replicates are available in Supplemental Figure S5. (D) Distribution of cell area growth for each time interval. Statistical analysis (multidimensional scaling) available in Supplemental Figure S6. Distribution of cell area growth related to the number of cell divisions in the lineage is available in Supplemental Figure S7.

Figure 6:. Cells can have regions with different growth rates in both WT and LGOoe.

(A-B) 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 WT (A) and LGOoe (B). 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. Representative images from n=3 biological replicates. Additional replicates are available in Supplemental Figure S8.

Figure 7:. Cell growth rate and direction averages to be uniform across the organ in WT, LGOoe, and lgo-2 but do not average in ftsh4–5, lgo-2 ftsh4–5, and LGOoe ftsh4–5.

(A-F) Heat maps from 24-hour time point to 96-hour time point for cell area growth (top rows) and ratio of proximal-distal cell growth to medial-lateral cell growth (bottom rows) projected on the earlier time point for WT (A), lgo-2 (B), LGOoe (C), ftsh4–5 (D), lgo-2 ftsh4–5 (E), and LGOoe ftsh4–5 (F). Three replicates are shown for each genotype, and each replicate has heat maps of both measures. The scale bar is 50μm. The principal directions of cell growth are overlaid on the heat maps as black lines that are oriented in the direction that each cell had the most growth and have a length that corresponds to the magnitude of the ratio of growth parallel to the principal direction of growth to the growth perpendicular to the principal direction of growth. Top rows: 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 10. Bottom rows: A proximal distal axis was defined, and the heat map represents the ratio of cell growth parallel to the axis divided by perpendicular to the axis. The lowest heat map value is 1, which represents equal amounts of growth along both axes. The highest heap map value is 1.7, which represents 1.7x more proximal-distal growth than medial-lateral growth. Red circles mark the region of the sepal with greater area growth (top rows) and greater proportion of proximal-distal growth (bottom rows). Quantification of the relationship between growth rate and proximal distal growth orientation is available in Supplemental Figure S9.