Fig. S4.

Constitutive PSBS expression suppresses the reduced qE phenotype of the npq4 mutant after UV-B exposure. (A) Sequence of the PSBS1 protein with the transit peptide predicted by the PredAlgo algorithm shown in red. It should be noted that the two paralogous genes PSBS.1 and PSBS.2 of C. reinhardtii encode proteins that differ in only one residue (at position 28). (B) Synthetic sequence of the psbS transgene excluding the predicted transit peptide, optimized for the codon use of the C. reinhardtii chloroplast. (C) Map of the transgene inserted in the inverted repeat of the chloroplast genome. Expression of the psbS sequence is driven by the psaA promoter/5′ UTR and by the rbcL 3′ UTR. The selection marker atpA::aadA::rbcL confers resistance to spectinomycin. The primers used for genotyping (AG1, AG2, and AG3) are indicated by arrows below the map. (D) Genotyping of psaA::psbS transformants in the npq4 nuclear background. DNA extracts from eight independent transformants (nos. 1–8) served as template for PCR amplification with the primers AG1/AG2 or AG1/AG3 (the products are loaded in alternate lanes of the agarose gel used for electrophoresis). AG1: 5′-GTC GTG GAG TAT TTA ATA CAG C-3′; AG2: ​5′-GAC TTG TTG GTA AAA CTG C-3′; AG3: 5′-GCT TTT GTT CCC TTT AGT G-3′. The PCR of the untransformed npq4 mutant is shown as a control. PCR of an 80-fold dilution of the npq4 template (npq4/80) indicates that even a single copy of the untransformed genome would have been detected had the transformants been heteroplasmic (each Chlamydomonas cell harbors a single chloroplast containing ∼80 copies of the chloroplast genome). This analysis shows that lines 1, 2, 4, 6, and 7 were homoplasmic. (E) Cultures of untransformed npq4 and psaA::psbS transformants in the npq4 background were grown in acetate-containing medium (Tris acetate phosphate) under dim white light (10 µmol⋅m⁻²⋅s⁻¹). Total proteins were extracted and analyzed by SDS/PAGE and immunoblotting with antisera against PSBS or tubulin as a control. (F and G) Immunodetection of PSBS (F) and LHCSR1 (G) in npq4 and psaA::psbS transformant no. 4 exposed to UV-B for 6 h. Increasing protein amounts of npq4/psaA::psbS (F) or npq4 (G) are loaded to show the linearity of the detection. CF1 (ATPase) is shown as a loading control. (H) qE values in npq4 (n = 6) and npq4/psaA::psbS strains (n = 5) after 6-h UV-B exposure. (I) Time course of qE induction in npq4 (n = 6) and npq4/psaA::psbS (n = 5) after 6 h UV-B exposure. (J) Cultures of npq4 and npq4/psaA::psbS (grown in minimal medium and not exposed to UV-B, because PSBS expression is constitutive in the latter strain) were photographed (t = 0), exposed to high light (1,000 µmol⋅m⁻²⋅s⁻¹) for 5 h, and photographed again (t = 5 h). Constitutive overexpression of PSBS leads to a delay in photobleaching. Data shown are representative of four independent biological repetitions.