Figure 5: BRI1 ubiquitination controls brassinosteroid signalling and BRI1 localization.

(a) Phenotypic analysis of 4-week-old bri1/BRI1–mCitrine and bri1/BRI1_25KR–mCitrine mono-insertional homozygous plants. A representative transgenic line is shown for both genotypes. (b) Average petiole lengths of the fourth true leaf from 4-week-old bri1/BRI1–mCitrine and bri1/BRI1_25KR–mCitrine plants. Experiments were carried out in triplicates. Error bars represent s.d. (n=20). The asterisk indicates a statistically significant difference between bri1/BRI1–mCit and the bri1/BRI1_25KR–mCit control (Mann–Whitney, P<0.0001). (c) Western blot analyses on bri1/BRI1–mCitrine and bri1/BRI1_25KR–mCitrine plants using anti-GFP and anti-BES1 antibodies. The asterisk indicates nonspecific signals used as loading control. (d) Quantification of dephosphorylated BES1 (left) and BRI1 (right) protein levels in bri/BRI1–mCit and bri1/BRI1_25KR–mCit. Experiments were carried out in triplicates. Error bars represent s.d. (n=3). The asterisk indicates a statistically significant difference in dephosphorylated BES1 and BRI1 levels between bri1/BRI1–mCit and bri1/BRI1_25KR–mCit (Mann–Whitney, P<0.05). (e) Confocal microscopy analyses of bri1/BRI1–mCitrine and bri1/BRI1_25KR–mCitrine roots. Scale bars=7 μm. (f) Quantification of the ratio between plasma membrane and intracellular fluorescence signal intensities of BRI1–mCitrine and BRI1_25KR–mCitrine. Experiments were carried out in triplicates. Error bars represent s.d. (n=15). The asterisk indicates a statistically significant difference between bri1/BRI1–mCit and the bri1/BRI1_25KR–mCit (Mann-Whitney, P<0.0005).