Figure 3.

Confocal time-lapse imaging over 1,760 min of an Arabidopsis seedling expressing H2B::mEosFP and developing collet and root hairs asymmetrically due to differences in hydration levels on opposite sides of the seedling. A to D, Maximum intensity projections of z stacks taken at 720 (A), 1,080 (B), 1,260 (C), 1,440 (D), and 1,760 (E) min to assess color changes in nuclei. Six nuclei labeled A to F (section A) followed over time; nuclei A, B, and C belong to cells that exhibited rapid polar elongation whereas D, E, and F belong to cells that did not differentiate further. E, Color change in nuclei A to F (section A) calculated as the green to red ratio of the respective mean fluorescence values (0–255 value scale) plotted over time informs about the start time and progression through for an endocycle. F, Measurement of green (G) and red (R) fluorescence values in two visible nuclei (arrows) in the last image in the time series (1,760 min; F) suggests clear differences in color. G, Nuclei (n = 96) from epidermal and collet or root hair cells observed in F classed on the basis of their green to red ratio presented as a bar diagram. A value close to 0 represents a red and a ratio close to 1 a more green color. When considered overall the nuclei fall into two populations on the basis of their color. One population centers on green/red 0.47, and the other, separated by a gap of 0.65, around 0.77. H, A color overlay representation reflects the distribution of the two groups of different-colored nuclei with values below 0.65 depicted in red and above 0.65 overlaid in green. The seedling is divided into two asymmetrically developing zones. I, A box plot representing the two groups of different-colored nuclei with values below 0.65 depicted in red and above 0.65 depicted in green. The comparison of both populations shows that nuclei with ratios above 0.65 tend to be larger than nuclei below this ratio (P value < 0.00001). J, Establishing correlations between color change (green to red ratio) due to net increase in H2B::mEosFP content in nuclei, alteration in planer nuclear area, and the total nuclear fluorescence. Pairwise two-dimensional scatter plots showing planar nucleus area (in μm²; J1), the total nuclear fluorescence (J2), and the green to red ratio suggest a positive correlation between the three parameters (R2 J1 = 0.460; J2 = 0.711; J3 = 0.582). K, A combined three-dimensional scatter plot and multiregression analysis (ANOVA R2 = 0.8459) reaffirms a strong correlation of changes in the green to red ratio to other parameters.