Skip to main content
. Author manuscript; available in PMC: 2020 Nov 25.
Published in final edited form as: Nat Plants. 2020 May 25;6(6):686–698. doi: 10.1038/s41477-020-0666-7

Extended Data Fig. 7. Cytokinin treatment mimics the drmy1–2 mutant.

Extended Data Fig. 7

a,b, Confocal imaging of the whole inflorescences of WT (a) and drmy1–2 (b) cultured in mock conditions or 5 μM BAP (synthetic cytokinin) for 6 days. p35S::mCitrine-RCI2A: gray, for plasma membrane. Phenotypes quantified in Extended Data Fig. 7c. Blue or red arrowheads: flowers with obvious delayed sepal initiation phenotypes. Scale bars: 50 μm.

c, Graph characterizing the proportions of flower phenotypes observed after 5 μM BAP treatment for 6 days. Normal phenotype (N, black) is defined as similar to wild type. Mildly affected phenotype (M, grey) is similar to drmy1–2. Severely affected phenotype (S, silver) is more severe than drmy1–2. n = 23 (N: 23/23, M: 0/23, S: 0/23) flowers from 7 inflorescences for mock treated wild type; n = 42 (N: 4/42, M: 35/42, S: 3/42) flowers from 12 inflorescences for mock treated drmy1–2; n = 47 (N: 2/47, M: 14/47, S: 31/47) flowers from 16 inflorescences for BAP treated wild type; and n = 37 (N: 0/37, M: 3/37, S: 34/37) flowers from 18 inflorescences for BAP treated drmy1–2. A one-sided Kolmogorov-Smirnov test was used to compare the distributions of different situations. p-value for WT mock versus drmy1–2 mock: 7.73 E-14; p-value for WT mock versus WT BAP: 1.67 E-16; p-value for WT BAP versus drmy1–2 mock: 4.53 E-9; p-value for WT BAP versus drmy1–2 BAP: 7.55 E-3; p-value for drmy1–2 mock versus drmy1–2 BAP: 1.95E-15.

d, Graph of flower radius to assess developmental stage based on flower size. The radii of flowers without sepal primordia (SP = 0) or with only the outer sepal primordium (SP = 1) were measured for wild-type and drmy1–2 inflorescences cultured in either mock or 5 μM BAP for 6 days. The critical size threshold for outer sepal initiation is specified with a yellow dashed line. Note this size is the same for wild type mock and wild type BAP samples, indicating the stage of outer sepal initiation is not affected by BAP treatment. In contrast, for BAP treated drmy1–2, this characteristic size is more variable, consistent with the strongly enhanced phenotype. In the violin plots, the black line represents the median and the individual data points are shown. WT Mock SP = 0: n = 5; WT Mock SP = 1: n = 12; drmy1–2 Mock SP = 0: n = 11; drmy1–2 Mock SP = 1: n = 14; WT BAP SP = 0: n = 19; WT BAP SP = 1: n = 19; drmy1–2 BAP SP = 0: n = 19; drmy1–2 BAP SP = 1: n = 19.

e, 5 μM BAP treatment on the DR5 auxin signaling reporter (white) for 3 days. p35S::mCherry-RCI2A: red, for plasma membrane; Red arrowhead: indicates the same flower before and after the BAP treatment. Scale bar: 50 μm. Note the DR5 signal becomes more diffuse after cytokinin treatment. n = 3 inflorescences.

f, 5 μM BAP treatment on PIN1-GFP (cyan) auxin efflux carrier for 2 days. Red arrowhead: indicates the same flower before and after the BAP treatment; Scale bar: 50 μm. PIN1-GFP appears to form additional convergence points similar to drmy1–2. n = 3 inflorescences.

g, Long-term treatment of flowers with 5 μM BAP causes severe sepal size defects. Wild-type inflorescences were cultured for 6 days on mock or BAP media, dissected to reveal flowers with initiating sepals, and further cultured for 14 days to examine the effects on sepal size. n = 3 flowers.

h, The sepal area distribution for mature WT, drmy1–2, ahp6, drmy1–2ahp6, and tir1–1afb1–1afb2–1afb3–1 (tir1afb1-2-3 for short) sepals. The boxes extend from the lower to upper quartile values of the data and the whiskers extend past 1.5 of the interquartile range. Outliers are indicated with small dots. Sepals from different flowers were pooled together. n = 35 flowers. Wild-type and drmy1–2 data was subsampled from that shown in Extended Data Figure 1b. Two-tailed Student’s t test * p-value < 0.05 (p-value for the mean of sepal area, WT versus drmy1–2: 2.70E-33; WT versus ahp6: 1.42E-38; WT versus drmy1–2ahp6: 9.91E-25; WT versus tir1abf1–1afb2–1afb3–1: 1.22E-39).

i, Average coefficient of variation (CV) calculated for the areas of all sepals in each single flower for WT, drmy1–2, ahp6, drmy1–2ahp6, and tir1–1afb1–1afb2–1afb3–1. n = 35 flowers. Two-tailed Student’s t test * p-value < 0.05 (p-value for the mean of CV, WT versus drmy1–2: 4.99E-14; WT versus ahp6: 1.75E-13; WT versus drmy1–2ahp6: 8.06E-16; WT versus tir1abf1–1afb2–1afb3–1: 3.31E-32). Measure of centre: mean. Error bars represent standard error of the mean.