Files in this Data Supplement:
Fig. S1. BRs control root meristem size. (A) Meristem length in Col-0 and bri1. DAG, days after germination. (B) Cortical cell number present in the apical, basal and whole meristem of 7-day-old wild-type and cpd seedlings. Data represent the mean ± s.e.m.
Fig. S2. The expression level of RCH1 is not affected by BRs. qRT-PCR expression analysis of 6-day-old Col-0 seedlings grown on (A) 0.5× MS, or (B) 0.5× MS supplemented with 2 µM BRZ. Seedlings with similar root length were treated with 100 nM BL for 3 and 16 hours. Data represent the mean ± s.e.m. of two independent experiments.
Fig. S3. The response of stem cell niche markers to low BR level. (A-F) Representative CLSM image showing the expression of fluorescently tagged stem cell niche markers in 6-day-old Col-0 seedlings treated with mock (left) or 3 µM BRZ (right). Cell borders are marked by PI (red). Scale bar: 20 µm. (B) The confocal image is presented as maximal projection.
Fig. S4. BRI1 is expressed throughout the root meristem. (A) CLSM image of pBRI1-BRI1-GFPox root. GFP signal is shown in green and PI in red. Scale bar: 50 µm. (B) Cell type-specific expression of BRI1 (At4g39400). Data is derived from Birnbaum et al. (Birnbaum et al., 2003) by Genevestigator (https://www.genevestigator.com/gv/index.jsp). Root cell files are marked as in Fig.1A.
Fig. S5. Expression pattern of the different BRI1-GFP transgenic lines. Transgenic lines were grown in the absence (left) and presence (right) of 3 µM BRZ. Maximal projection of confocal images showing (A) bri1;pGL2-BRI1-GFP, (B) bri1;pSCR-BRI1-GFP and (C) bri1;pSHR-BRI1-GFP roots. Note that in the presence of BRZ, BRI1-GFP shows a scrambled expression pattern in bri1;pGL2-BRI1-GFP (Kuppusamy et al., 2009). However, this phenotype is largely rescued under normal conditions. Scale bar: 50 µm.
Reference
Kuppusamy, K. T., Chen, A. Y. and Nemhauser, J. L. (2009). Steroids are required for epidermal cell fate establishment in Arabidopsis roots. Proc. Natl. Acad. Sci. USA 106, 8073-8076.
Fig. S6. BRL1 and BRL3 are not involved in epidermal control of root growth. Quantitative expression analysis of BRL1 and BRL3 in seedlings treated with BL for 3 hours and in seedlings grown on 2 µM BRZ for 6 days. Roots were taken for analysis. Expression level is normalized to Col-0 control. Data represent the mean ± s.e.m of two independent experiments. Note that the expression level of BRL1 and BRL3 is similar in the backround of the transgenic lines.
Fig. S7. pBRI-BRI1-GFPox is resistant to the inhibitory effect of BRZ as compared to wild type and bri1;pGL2-BRI1-GFP. Root length of 7-day-old seedlings. Data represent the mean ± s.e.m.
Fig. S8. BRI1 expression in the inner cell files does not promote or inhibit root length. (A,B) Root phenotype of bri1;pGL2-BRI1-GFP;pSCR-BRI1-GFP double-transgenic plants as compared with the parental bri1;pSCR-BRI1-GFP and bri1;pGL2-BRI1-GFP lines. CLSM image (A) and root length of 7-day-old seedlings (B). (C,D) Root phenotype of bri1;pGL2-BRI1-GFP; pSHR-BRI1-GFP double-transgenic lines as compared with the parental bri1;pSHR-BRI1-GFP and bri1;pGL2-BRI1-GFP lines. CLSM image (C) and root length of 7-day-old seedlings (D). Data shown in B and D represent the mean ± s.e.m. of three independent experiments. Scale bar: 50 µm.
Fig. S9. BRI1-mediated signal, from the epidermis to the inner cells, as opposed to local BR response. (A) CLSM image of pAGL42-GFP in 7-day-old Col-0 seedlings treated with mock (left) or 3 µM BRZ (right). Note that the expression of the QC marker pAGL42-GFP can extend to the stele, in agreement with the cell type-specific expression data (Fig. 6B,C). (B) CLSM image of pGL2-BES1D-GFP seedlings treated with mock (left) or 100 nM BL (right). Cell borders are marked by PI (red). Scale bar: 20 µm (C) Cross-section of bri1 expressing pDWF4-GUS. Note the epidermal expression of the marker in the meristem. Scale bar: 20 µm.
