Figure 1.

Characterization of an insertional CLiP mutant of C. reinhardtii (Crvtc2-1; LMJ.RY0402.058624) affected in the VTC2 gene that encodes GDP-l-Gal phosphorylase. A, Physical map of VTC2 (obtained from Phytozome, version 12.1.6) with the CIB1 cassette insertion site in the Crvtc2-1 mutant. Exons are shown in black, introns in light gray, and promoter/5′ UTR and terminator sequences in dark gray. The insertion site of the CIB1 cassette is indicated by the triangle, and the binding sites of the primers used for genotyping and gene expression analysis of Crvtc2-1 are shown as black arrows. The sequence encoding the catalytic site of GDP-l-Gal phosphorylase is marked as a white line within Exon 3. B, PCR performed using primers annealing upstream of the predicted cassette insertion site in VTC2 (top, primers P1+P2) and primers amplifying the 5′ and 3′ genome-cassette junctions (middle and bottom; P3+P4 and P5+P6, respectively). The expected sizes are marked with arrows. C, Ascorbate contents of the wild type (CC-4533) and the Crvtc2-1 mutant grown mixotrophically in TAP medium at moderate light with and without the addition of 1.5 mm H₂O₂. D, Transcript levels of VTC2, as determined by RT-qPCR in cultures supplemented, or not, with H₂O₂ using primers P7 and P8. E, Electrophoresis of RT-PCR products using primers P9 and P10, spanning the sequence that encodes the catalytic site of GDP-l-Gal phosphorylase. The number of PCR cycles is indicated at the bottom of the figure. The presented data are based on three independent experiments. When applicable, averages and ses (±se) were calculated. Data were analyzed by one-way ANOVA followed by Dunnett’s posttest: ×, P < 0.05, ××, P < 0.01, and ××××, P < 0.0001, compared to the untreated CC-4533 strain.