Isolation of cp31a, cp31b, and a/b double mutants. (A) Schematic representation of gene organization and location of T-DNA insertions in CP31A (Left) and CP31B (Right) genes (drawn to scale). Insertion sites of T-DNAs were determined by sequencing PCR products generated with a left border (LB) and a gene-specific primer. The T-DNA in cp31a-1 plants is inserted before base pair 1530 and that in cp31a-3 plants is inserted before base pair 542. For line cp31b-4, we found a back-to-back insertion of 2 T-DNAs at positions 1036 and 1045, respectively. Gene-specific 10-mers adjacent to LB are shown for each T-DNA. Black, chloroplast targeting peptide; striped, RRM domains 1 (vertical striping) and 2 (horizontal striping); white, acidic domain; gray, spacer; triangles, insertion sites for T-DNAs. Numbers refer to positions relative to the ATG (A = 1). Solid horizontal lines represent introns. (B) Loss of CP31A and CP31B proteins in cp31a and cp31b single and double mutants. Immunoblot analysis of CP31A/B accumulation in mutant and WT tissue. Equal amounts or indicated dilutions of WT Col-0 total leaf proteins were analyzed by probing immunoblots with anti-tobacco CP31 antisera. Observed sizes match in silico predictions after removal of signal peptides and those determined previously by organelle import for CP31A (8). The same filter was stained with Ponceau S to visualize RbcL, the large subunit of Rubisco, as loading control. (C) CP31A and CP31B mRNA is undetectable in mutants. RT-PCR was used to assay mRNA levels in cp31a or cp31b mutants, respectively, using primer combinations cp31.ex4.for/cp31.ex7.rev and cp31B.revT/cp31B.for. The bottom panel shows RT-PCR amplification of PPR54 mRNA in the same RNA samples (PARAex1for/PARAex2rev). Comparison of DNA-based amplification products (lane DNA Col-0) with cDNA-derived amplification products shows that no intron-containing amplification products were detected, demonstrating that the bands observed arose from RNA templates. Expected sizes: CP31B DNA = 429 bp, cDNA = 332 bp; CP31B DNA = 372 bp, cDNA = 285 bp; PPR54 DNA = 322 bp, cDNA = 402 bp. (D) Phenotypes of cp31a-, cp31b- and double-mutant seedlings grown on soil in growth chambers.

RNA gel blot hybridizations showing plastid RNA accumulation for selected genes in cp31 mutants. Total leaf RNA was fractionated on 1.2% agarose gels and analyzed by hybridization to probes for the plastid RNAs indicated. Equal loading was controlled by subsequently hybridizing filters to a probe for 16S rRNA and/or by staining the cytosolic 25S rRNA (25S) with methylene blue. Note that the filter probed for ndhF was subsequently also probed for psbA, which, together with the methylene blue stain of the cytosolic 25S rRNA, provides 2 internal loading controls. In the case of rps14, the tricistronic psaA/psaB/rps14 mRNA is shown, because we were unable to detect a monocistronic rps14 mRNA.

Levels of plastid transcripts in cp31 mutants. All chloroplast mRNA and rRNA transcripts were measured from cp31 mutants and WT plants by qRT-PCR. The graph depicts the log₂ ratio of transcript levels in the mutants compared with levels in the WT plants. The strongest decrease in transcript level was seen for the ndhF mRNA. The genes are sorted according to their physical location on the chloroplast chromosome. The full data set is given in Table S3.

Monitoring of NDH activity by chlorophyll a fluorescence analysis. (Upper) The curve shows a typical trace of chlorophyll a fluorescence in WT seedlings (WT: Col-0). Leaves were exposed to actinic light (AL; 50 μmol photons m⁻² s⁻¹) for 5 min. AL was turned off, and the subsequent change in fluorescence level was monitored. (Lower) The postillumination chlorophyll a fluorescence curve is a characteristic feature of NDH activity, and was therefore magnified from the boxed area for all genotypes analyzed. ML, measuring light.