Supplemental Data. Ohnishi et al. Plant Cell. (2010) /tpc anti-lci1 NH 4+ NO 3- DAPI IF DAPI IF

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1 Supplemental Data. Ohnishi et al. Plant Cell. (2010) /tpc A DAPI HC IF DAPI LC IF antilci1 B DAPI IF DAPI IF C2 E4 Supplemental Figure 1. Indirect immunofluorescence analyses of LCI1 protein in Chlamydomonas cells. The subcellular localization of LCI1 protein was analyzed by an indirect immunofluorescence method using affinitypurified LCI1 antibody. Indirect immunofluorescence of LCI1 in photosynthetically wildtype strain Q304P3 grown in either HC or LC conditions (A) and in the LCI1 transformants C2 and E4 (B) grown in either HSM(NH4) or HSM(NO3) under HC conditions. White arrows show the position of nuclei. DAPI, DAPI staining. IF, immunofluorescence. Scale bars, 5 µm.

2 Supplemental Data. Ohnishi et al. Plant Cell. (2010) /tpc (1) (2) (3) (4) Supplemental Figure 2. Comparison of amino acid sequences between LCI1 and proteins that contain similar domain structure to LCI1. Amino acid sequences of LCI1related proteins from Chlamydomonas reinhardtii (), Volvox carteri (), Chlorela sp. NC64A (), Coccomyxa sp. C169 (), Saccharomyces cerevisiae (), and Yarrowia lipolytica () were aligned by using a program MAFFT version 6 ( with a strategy of EINSi (Katoh et al., 2005). Identical and conserved amino acids were shown in red and blue, respectively. Numbered bars on the sequences show hydrophobic regions of LCI1. Amino acid sequence data in this figure can be found in the EMBL/GenBank/DDBJ data libraries under accession numbers EDP06234 (A flagellar associated protein FAP292 in ) and AAC41664 (A putative osmosensor SHO1 in ), or in the JGI database ( under protein ID numbers shown in the left column.

3 Supplemental Data. Ohnishi et al. Plant Cell. (2010) /tpc LCI CST Supplemental Figure 3. Hydropathy profiles of LCI1 and CST1. The profiles were determined by the method of Kyte and Doolittle (1982) with a window size of 17 amino acids by using a program GENETYXMAC ver

4 Supplemental Table 1. Ci affinity of the photosynthetically WT Q304P3, the lcr1 mutant and the LCI1 transformants at ph 7.0. Strain CO 2 condition N source Vmax (µmol O 2 mgchl 1 h 1 ) K 1/2 (µm) Q304P ± ± ± ± 3 lcr ± ± ± ± ± ± ± ± 4 C ± ± ± ± ± ± ± ± 5 E ± ± ± ± ± ± ± ± 5 The data are shown with ±SD, which were obtained from three independent experiments.

5 Supplemental Table 2. Ci affinity of the photosynthetically WT Q304P3, the lcr1 mutant and the LCI1 transformants at ph 7.8. Strain CO 2 condition N source Vmax (µmol O 2 mgchl 1 h 1 ) K 1/2 (µm) Q304P ± ± ± ± 5 lcr ± ± ± ± ± ± ± ± 8 C ± ± ± ± ± ± ± ± 5 E ± ± ± ± ± ± ± ± 8 The data are shown with ±SD, which were obtained from three independent experiments.

6 Supplemental Methods RNA gel blot analysis Total RNA of Chlamydomonas cells was isolated using an RNeasy Plant Mini Kit (QIAGEN GmbH, Hilden, Germany). Five µg of total RNA was separated on a denaturing 1.0% agarose gel at 5 V cm 1 and then blotted onto a nylon membrane. The blots were hybridized with a cdna probe specific to the mrna of Lci1. The cdna probe corresponding to the coding region of Lci1was prepared by amplifying the cdna fragment using the primer set described in the methods and total cdna that had been prepared using SuperScript III (Invitrogen Co. Carlsbad, CA, USA). The cdna probe amounting to 10 pmol µl 1 was labeled with [α 32 P]dCTP (500 µci ml 1 ) using a DNA random labeling kit (TaKaRa Bio. Inc., Shiga, Japan). Hybridization was performed for 12 to 18 h at 65 C using Church phosphate buffer (Church and Gilbert, 1984). The signals were detected by autoradiography using a fluorescentimage analyzer (FLA3000; FUJIFILM, Tokyo, Japan). Indirect immunofluorescence analysis Cells in logarithmic growth phase (OD730= ) were collected by centrifugation at 700 x g and then resuspended in PBS containing 0.1% Tween 20 (w/v) (PBST) at a concentration of OD730=1.0 to 1.5. Forty µl of cell suspension was spotted onto PolyPrep Slides (SigmaAldrich Co.) and then left at room temperature for 5 min. The spotted cells were washed twice with PBST (5 min each) and subsequently fixed with 100 µl of 8% paraformaldehyde (w/v) for 15 min at room temperature. The paraformaldehydefixed cells were washed three times with PBST (5 min each) and kept in icecold methanol for 30 min at 30 C. Subsequently, the slide glasses with fixed cells were dried at room temperature for 23 min and kept in PBST for more than 10 min. Then, 100 µl of blocking solution (PBS buffer containing 5% BSA (w/v)) was spotted onto the cells and incubated at room temperature for 1 h. After three washes with PBST (5 min each), 100 µl of PBST containing 1 x 10 2 volume of affinitypurified antibody against LCI1 was spotted and incubated at 37 C for 1.5 h. Subsequently, the cells were washed six times with PBST (5 min each) and 100 µl of PBST containing 2.55 x 10 2 volume of AlexaFluor 488conjugated antirabbit IgG antibody (Invitrogen Co. Carlsbad, CA, USA) was spotted, which was then incubated at 37 C for 1.5 h. The cells were washed six times with PBST (5 min each). Ten µl of PBS containing 10 µg ml 1 of DAPI was spotted onto the cells and then the cells were observed with a Zeiss Axiophot microscope (Carl Zeiss, Oberkochen, Germany) with a filter set containing excitation filter D450480, dichroic mirror 425DCLP, and emission filter D (Chroma Technology Corp., Rockingham, VT, USA). The fluorescent images were captured using a cooled chargecoupled device camera VB7010 (KEYENCE, Osaka, Japan). To visualize DNA in the cell, fixed cells stained with 10 µg ml 1 of DAPI and observed using an Axiophot microscope with a filter set including exciter D360/40, dichroic 400DCLP, and emitter D460/75 (Chroma Technology Corp).

7 Supplemental References: Church, G.M., and Gilbert, W. (1984). Genomic sequencing. Proc. Natl. Acad. Sci. USA 81: Katoh, K., Kuma, K., Toh, H., and Miyata, T. (2005). MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Res. 33: Kyte, J., and Doolittle, R.F. (1982). A simple method for displaying the hydropathic character of a protein. J Mol Biol 157: