Effect of temperature on fertility in wild type and cenh3-4 plants.

(A) Analysis of silique length through the main stem of wild type (WT) grown at 16°C (n=18), 21°C (n=25), 26°C (n=13), 30°C (n=20) and cenh3-4 mutant at 16°C (n=22), 21°C (n=20), 26°C (n=22) and 30°C (n=11). The silique position is numbered from the oldest to the youngest silique on the main stem. Error bars depict standard deviation. (B) Anthers of the abovementioned plants after Alexander staining. (C) Quantification of viable pollen per anther (n=45). Significance of the difference is counted using Two-tailed t-test.

Effect of temperature on meiosis duration and micronuclei formation.

(A) Graphical representation of the duration of Meiosis I (from the end of diakinesis to the end of anaphase I; Figure S2), Interkinesis, and Meiosis II (prometaphase II to telophase II) calculated from live imaging of anthers in wild type (WT) and cenh3-4 plants grown at 16, 21, 26 and 30°C. Error bars represent standard deviation (from 16 to 30°C: in wild type n=36,36, 36,46 and cenh3-4 n=35,24,24,4, resp.) Significance of the difference is indicated (two tailed t-test; **** p<0.0001). (B) Anther loculi in the tetrad stage of wild type, cenh3-4 and spo11-2-3 plants grown at 16, 21, 26 and 30°C. DNA was stained with DAPI. Blue arrowheads indicate examples of produced micronuclei. Scale bar=10 µm. (C) Number of micronuclei per lobe in wild type (WT, n=19,19,19,19), cenh3-4 (n=19,19,19,19), and spo11-2-3 (n=19,26,21) plants. Error bars represent standard deviation. Significance of the difference from plants of the corresponding genotype grown at 21°C is indicated (two tailed t-test; * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).

Effect of high temperature on centromere structure in wild type plants.

(A) eYFP:CENH3 expression and DAPI staining of meiotic pachytene (blue dots) and mitotic tapetal cells (red dots) in wild type plants grown at 21, 26 and 30°C. Scale bar=5µm. (B) quantification of CENH3 fluorescence intensity per centromere in pachytene. Each interleaved scatter plot with median and interquartile range shows results of an independent experiment (left graph n=102,103,125, middle n=359,220,233 and right graph n=286,268,233). (C) Quantification of eYFP:CENH3 signal intensity in tapetum cells of plants grown at 21, 26 and 30°C. Each graph represents an independent experiment (left graph n=369,262,233 and right graph n=358,266,213). (D) Expression of BMF1:eYFP in pachytene in plants grown at 21, 26 and 30°C. DNA is counterstained with DAPI. Scale bar=5µm. (E) Quantification of BMF1:eYFP signal intensity per centromere in pachytene in two independent experiments; left graph n=120 and right n=100. Two-tailed t-test is used to depict the significance of the difference.

Effect of high temperature on the duration of BMF3:GFP localization during wild type meiosis.

(A) Time lapse series of BMF3:GFP (cyan) and TagRFP:TUB4 (magenta) in PMC from nuclear envelope breakdown to telophase II. Scale bar=5µm. (B) Duration of BMF3:GFP signal in plants grown at 21°C (n=24), 26°C (n=32) and 30°C (n=31). Significance of the difference was calculated via Two-tailed t-test.