Fig. 4.

Suppression of plastoquinol synthesis. a Target regions of the RNAi experiment. A closed box shows the coding region of the homogentisate solanesyltransferase (HST) transcripts. Gray boxes show regions targeted by double-strand RNAs HST1-3. b RT-PCR assays for the transcripts of HST and actin. Lanes 1–3 (controls): RT-PCRs with total RNAs extracted from each of three cell suspensions for which electroporation was conducted without double-strand RNAs. Lanes 4–6: RT-PCRs with total RNAs extracted from each of three cell suspensions for which electroporation was conducted with HST1. RNA was extracted from cell suspensions 1.5 days after the electroporation. c Relative amount of plastoquinone (PQ) pool. Relative amount of quinones are evaluated as peak areas of MRM chromatogram in the LC-MS/MS analyses. Normalization of a PQ peak area was performed by a peak area of ubiquinone (UQ). Quinones were extracted from cells 2 days after the electroporation with or without HST1. All the reduced quinones, plastoquinol and ubiquinol, were oxidized with FeCl₃ prior to the LC-MS/MS analyses, resulting in PQ and UQ. Error bar shows standard deviation. The numbers on the bars show mean values. *: P < 0.05 (Welch t-test). N = 3. d Growth of chlamydomonad sp. NrCl902 until 2 days after the electroporation. Error bar shows standard deviation. N = 3. e RT-PCR assays for the transcripts of HST and actin in RNAi experiments with additional double-strand RNAs. Lanes 1–3 (controls): RT-PCRs with total RNAs extracted from each of three cells suspensions for which electroporation was conducted without double-strand RNAs. Lanes 4–6: RT-PCRs with total RNAs extracted from each of three cells suspensions for which electroporation was conducted with HST1. Lanes 7–9: RT-PCRs with total RNAs extracted from each of three cells suspensions for which electroporation was conducted with HST2. Lanes 10–12: RT-PCRs with total RNAs extracted from each of three cell suspensions for which electroporation was conducted with HST3. RNA was extracted from cell suspensions 1.5 days after electroporation. f Growth of chlamydomonad sp. NrCl902 until 3 days after electroporations. Error bar shows standard deviation. N = 3. RT-PCRs were performed 36 and 72 hours after electroporation (highlighted in grey). g RT-PCR assays for the transcripts of HST and actin in RNAi experiments after 3 days from electroporation. Lanes 1–3 (controls): RT-PCRs with total RNAs extracted from each of three cell suspensions for which electroporation was conducted without double-strand RNAs. Lanes 4–6: RT-PCRs with total RNAs extracted from each of three cell suspensions for which electroporation was conducted with HST1. Lanes 7–9: RT-PCRs with total RNAs extracted from each of three cell suspensions for which electroporation was conducted with HST2. Lanes 10–12: RT-PCRs with total RNAs extracted from each of three cell suspensions for which electroporation was conducted with HST3. RNA was extracted from cell suspensions 3 days after electroporation