Fig. 2. rcb-101/hmr-22 restores PIF4 stability and activity.

a Immunoblot analysis showing HMR and PIF4 levels in Col-0, hmr-22, rcb-101/hmr-22, and pif4-2 seedlings grown in 50 μmol m⁻² s⁻¹ R light for 96 h at either 21 or 27 °C. b qRT-PCR analysis of the steady-state transcript levels of YUC8, IAA19, and IAA29 in Col-0, hmr-22, rcb-101/hmr-22, and pif4-2 seedlings grown in 50 μmol m⁻² s⁻¹ R light for 96 h at 27 °C. Different letters denote statistically significant differences in the transcript levels (ANOVA, Tukey’s HSD, p < 0.01, n = 3 biological replicates). c Immunoblot analysis of HMR and PIF4 levels in Col-0, hmr-22, rcb-101/hmr-22, and pif4-2 seedlings during the 21 to 27 °C transition. Seedlings were grown in 50 μmol m⁻² s⁻¹ R light for 96 h and then transferred to 27 °C in the same light condition. Samples were collected and analyzed at the indicated time points. For (a, c) RPN6 was used as a loading control. The relative levels of HMR and PIF4, normalized to RPN6, are shown underneath the respective immunoblots. The asterisks indicate non-specific bands. The immunoblot experiments were independently repeated at least three times, and the results of one representative experiment are shown. d qRT-PCR analysis of the steady-state transcript levels of YUC8, IAA19, and IAA29 in Col-0, hmr-22, rcb-101/hmr-22, and pif4-2 during the 21 to 27 °C transition. Seedlings were grown as described in (c), samples were taken before (light gray) and 24 h after (dark gray) the 27 °C treatment. Fold changes in the transcript levels after the 27 °C treatment are shown above the columns. For (b, d), transcript levels were calculated relative to those of PP2A. Error bars represent the s.d. of three biological replicates. The centers of the error bars represent the mean values. The source data of the immunoblots in (a, c) and the qRT-PCR data in (b, d) are provided in the Source Data file.