Figure 2.

Complementation of the insertional CLiP mutant LMJ.RY0402.058624 (Crvtc2-1) affected in the VTC2 gene with the coding sequence of VTC2. A, Physical map of the Crvtc2-1+VTC2 plasmid containing the coding sequence of VTC2, the constitutive promoter PsaD, and the APH7″ resistance gene and the terminators TPSAD and TRBCS2. Exons are shown in black and promoter/5′ UTR terminator sequences in dark gray, and the sequence encoding the catalytic site of GDP-l-Gal phosphorylase is marked as a white line. The binding sites of the primers used are shown as black arrows. B, PCR performed using primers annealing in the promoter and VTC2 exon 1 (P11+P12). The expected size is marked with an arrow. C, Electrophoresis of RT-PCR products obtained using primers annealing to the sequence encoding the catalytic site of VTC2 (P9+P10). The expected size is marked with an arrow. D, Ascorbate contents of CC-4533, the Crvtc2-1 mutant, and the complementation lines Crvtc2-1+VTC2 grown for 3 d in TAP at 100 µmol photons m⁻² s⁻¹. The presented data are based on four independent experiments. When applicable, averages and ses (±se) were calculated. Data were analyzed by one-way ANOVA followed by Dunnett’s post-test: ××, P < 0.05 and ××××, P < 0.0001, compared to the CC-4533 strain. µE, µmol photons m⁻² s⁻¹.