The C-terminal half and coiled coil domain are essential for TOT3 functions. A) Schematic of TOT3 protein domains and motifs. B) Y2H assay between TOT3 lacking the C-terminus (1-296) and the coiled coil motif (1-610) and TOI5. The experiment was done in 3 replicates with same results. C) Co-IP between TOT3 lacking the C-terminus (1-296) and the coiled coil motif (1-610) and TOI5. The experiment was done in 2 replicates with similar results. D) Localization of TOT3 and TOT3 lacking the C-terminus (1-296) or the coiled coil motif (1-610) fused to GFP in the primary root meristem and transition zone. The inset shows TOT3 localization in 2 cells. The numbers indicate the number of seedlings corresponding to the representative localization out of total number of seedlings analyzed. Scale bar, 20 µm. Green, GFP; magenta, propidium iodide. Representative pictures E) and hypocotyl length quantification for truncated TOT3 constructs in tot3-2 at 21 and 28 °C in short-day conditions F). Scale bar, 5 mm. Box plots show median with Tukey-based whiskers and outliers. Center line, median; box limits, upper and lower quartiles; Tukey whiskers (1.5× interquartile range, the 25th percentile minus 1.5× interquartile range, the interquartile range is the difference between the 25th and 75th percentiles); points, outliers. The number of individually measured seedlings (n) from 2 independent replicate experiments is indicated above the x-axis. Letters indicate significant differences based on 2-way ANOVA and Tukey's test (P < 0.01) F). P-value is shown at the top. -WT, without Leu and Trp; -WTH, without Leu, Trp and His; IP, immunoprecipitation; IB, immunoblot.