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Figure S1. The BIT1 GFP line exhibits a hypersensitive response to red and far-red light. Hypocotyl growth response of wild type, BIT1 GFP-2, phyA or phyB seedlings under various intensities of red (R) and far-red (FR) light. The seedlings were grown for 5 days. Data are shown as means (n = 20) ± s.d.

Figure S2. The BIT1 knock-out line exhibits a hyposensitive response to blue light. (a) Hypocotyl growth responses of wild type (Col and WS) and rax2-1/bit1-1 under various intensities of blue light. Data are shown as means (n = 20) ± s.d.

The rax2-1/bit1-1 mutation is in a WS background. When we compared the blue light responses between WS and Col, we found that WS exhibited a lower blue light sensitivity than Col, under our experimental conditions. A lower blue light sensitivity was also observed for the WS ecotype in other experiments (Babourina et al., 2002).

(b) Anthocyanin contents of wild type and rax2-1/bit1-1 seedlings grown for 6 days under continuous blue light (20 μmol m−2 s). The measured anthocyanin contents (A535–A650/g fresh weight) are shown as means (n = 3) ± s.d.

Figure S3. BIT1 is not stabilized by red light. Mesophyll protoplasts from wild type plants were transfected with the BIT1-HA fusion construct and incubated overnight with a low concentration (1 μM) of MG132 in darkness. Cells were treated with 100 μM of cycloheximide and transferred to either red (15 μmol m−2s) or blue (10 μmol m−2s) light. Cells were harvested at the times indicated for extraction of total cellular protein. BIT1-HA proteins were detected by Western blotting using anti-HA antibody. Coomassie Brilliant Blue-stained RbcS protein (RBC) served as a protein loading control.

Figure S4. Negative controls in the bimolecular fluorescence complementation (BiFC) assay. YFPN-BIT1/YFPC and YFPN/YFPC-COP1 plasmids were cotransfected into mesophyll protoplasts. The BiFC signal was not detected. DAPI staining is observed, along with the DIC image. Scale bars, 10 μm.

Figure S5. Formation of nuclear speckles between BIT1-GFP and COP1 in Arabidopsis protoplasts. Following transfection of mesophyll protoplasts, BIT1-GFP was expressed alone (upper panel) or coexpressed with COP1-HA (lower panel) and then incubated in darkness. From left to light, GFP, DAPI and DIC images, respectively; a close-up nuclear image is shown in the white box. In the lower panel, nuclear speckles (arrowheads) were observed among a uniform green fluorescent background in the nucleus. Scale bars, 10 μm.

Figure S6. Nuclear localization of BIT1-GFP in transgenic lines. Hypocotyls from GFP (upper panel) and BIT1-GFP (lower panel) overexpression lines. Seedlings were grown for 5 days in darkness and then transferred to blue light (10 μmol m−2s) for 2 h. From left to light, GFP, DAPI and DIC images, respectively. In contrast to the GFP control, green fluorescence from BIT1-GFP was observed only in nucleus. Arrowheads indicate the nucleus. Scale bars, 10 μm.

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TPJ_3508_sm_figs.pdf501KSupporting info item
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