FIGURE 10.
The increase of γ-tocopherol content is dependent on CRR1. A, for immuno-detection of plastocyanin and cytochrome c6, soluble proteins were separated by SDS-polyacrylamide gel electrophoresis (15%) and transferred to a nitrocellulose membrane (0.1 n pore size). The abundance of OEE1 was used as a loading control (bottom panel). B and C, abundance of CYC6 (B) and HPPD1 (C) transcripts. Independent cultures corresponding to CRR1 (black circles), crr1 (triangles apex up), or crr1-ΔCys (triangles apex down) were grown in copper-replete (black symbols) or copper-depleted (white symbols) TAP medium. Cells were collected after reaching a density of 2–6 × 106 cells/ml and analyzed for RNA abundance. Each symbol represents an independent experiment analyzed in technical triplicates. D, the tocopherol content in Chlamydomonas crr1 and corresponding strains that have been complemented with full-length CRR1 (crr1(CRR1)) or with a truncated version of CRR1 lacking a cysteine-rich domain (crr1-ΔCys) was measured as described under “Experimental Procedures.” Data are the averages of three biological replicates ± S.D.