SUPPLEMENTARY INFORMATION

doi: 10.1038/nature05618

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Figure S1 | CPD DWF4 and BRI1 expression patterns in wild type Arabidopsis , (a-c), pCPD:GUS expression pattern in the developing shoot indicate that CPD isexpressed in all 3 layers: the epidermis, mesophyll and vasculature. (b) pCPD::GUSexpression is reduced when seedlings were grown on medium containing brassinolide (BL, the most active BR), indicating that the CPD promoter fragment contains BR responsive elements, as was previously reported 1. (c), A cross section in pCPD:GUS leaf primordia. (d), pDWF4::GUS expression pattern (DWF4 encodes an upstream enzyme in the BR biosynthesis pathway). A cross section through the shoot apical meristem of pDWF4::GUS line shows that the GUS signal was found in

both the outer and inner layers of the meristem and young leaf primordia. The GUS signal is less intense in the older parts of the leaf. (e-h), In situ hybridization with BRI1 anti-sense (e,g) and sense (f,h) probes. Shoot apical meristem (e,f) and floral meristem (g,h) are shown. Note that BRI1 is expressed in both outer and inner layers of young primordia. (i-k), Confocal microscope analysis of a pBRI::BRI1:GFP line2. BRI1:GFP signal (green) is detected in both the mesophyll (d) and epidermal (e and * in d) cells of a young leaf and in the outer and inner cell layers of a floral bud (f). Auto-fluorescence is detected as a red signal.

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Figure S2 | ATHB8::GUS expression pattern and western blot of bri1;AtML1::bes1-d:GFP. (a), ATHB8::GUS expression pattern. To verify the specificity of the promoter at high sensitivity, we transformed plants with pATHB8::GUS and stained whole seedlings from 15 independent transformants. In our hands, the ATHB8 promoter is expressed in the vasculature, but many lines also had a diffuse expression pattern

outside the vasculature tissues The GUS signal within and outside the vaculature is indicated by an arrow and an arrowhead respectively. (b), Total protein extract from either WT or bri1;AtML1::bes1- d:GFP seedlings were probes with anti-GFP antibodies. Note that bes1-d:GFP is migrating as a single band.

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doi: 10.1038/nature05618 SUPPLEMENTARY INFORMATION

METHODSIn situ hybridization and western blot BRI1 mRNA was detected with digoxigenin-labelled riboprobes using the method found at http://www.its.caltech.edu/~plantlab/protocols/insitu.htm. The entire coding region was used for both anti-sense or sense probes. Western blot against bes1-d::GFP was performed using anti-GFP antibodies (GFP (B2), Santa Cruz biotechnology) Promoter::GUS constructs and analysis The GUSPlus gene was amplified from CAMBIA1305.1 as a SmaI/BamHI fragment and

was cloned in pBJ36. For pCPD::GUS and pDWF4::GUS, 968 bp and 1127 bp genomic sequence upstream of the predicted coding sequences were amplified from genomic DNA and were cloned as BamHI/BamHI and PstI/KpnI respectively in pBJ36/GUS. Final constructs were introduced into the binary vector pMLBART and transformed into wildtype Columbia.

26. Mathur, J. et al. Transcription of the Arabidopsis CPD gene, encoding a steroidogenic cytochrome P450, is negatively controlled by brassinosteroids. Plant J 14, 593-602 (1998).

27. Friedrichsen, D. M., Joazeiro, C. A., Li, J., Hunter, T. & Chory, J. Brassinosteroid-insensitive-1 is a ubiquitously expressed leucine-rich repeat receptor serine/threonine kinase. Plant Physiol

123, 1247-56 (2000).