Fig. 6: The not4a mutant mimics the pgr3 pentatricopeptide mutant.
a Volcano plot of GO chloroplast-associated (GO:0009507) DEGs in not4a vs WT. The top 8 DEGs are labelled (UP: ELIP2/AT4G14690, ELIP1/AT3G22840, PGL5/AT5G24420 and DOWN: AT3G55240, AT5G24240, SAUR1/AT4G34770, PLP2/AT2G26560, AT4G16620). b Quantitative PCR (qPCR) of PGR3 in Col-0, not4a and N4A-G3 complementation line. Expression levels normalised to ACTIN7 and shown relative to Col-0 WT. Error bars = ±SEM. n = 3 biological replicates. Letters indicate one-way ANOVA; Tukey’s test (p < 0.01). c Representative rosette images of 4-week-old Col-0, not4a, Ws-4 and pgr3-4 lines grown under long day (LD) conditions. Bar = 1 cm. d 10-day-old Col-0, not4a, Ws-4 and pgr3-4 seedlings grown on vertical and horizontal plates ±10 µM lincomycin. Bar = 1 cm. e Western blot determination of 30 S ribosome subunits (RPS1 and RPS7), 50 S ribosome subunits (RPL2 and RPL4), PetC and NdhH in Col-0, not4a, Ws-4 and pgr3-4 with coomassie blue (CBB) staining to show comparable protein loading. Blots were repeated at least three times with similar results. f qPCR of PetL and PetG in 10-day old Col-0, not4a, Ws-4 and pgr3-4 seedlings. Expression levels normalised to ACTIN7 and shown relative to Col-0 WT. Data are average of three biological replicates. Letters indicate one-way ANOVA; Tukey’s test (p < 0.01). Error bars = ±SEM. g Relative amounts (ng/μl) of 50 S (23 s and 23 sb) and 30 S (16 s) plastid rRNAs in Col-0, not4a, Ws-4 and pgr3-4 seedlings. Values are quantified from Tapestation data shown in Supplementary Fig. 5, following normalisation to cytosolic 18 s rRNA. Col-0 and not4a data are same as in Fig. 3e. Error bars = ±SEM. n = 3 biological replicates. *p = 0.0446, **p = 0.0037, ***p = 0.027, students t-test, two-tailed. h ETR(I) and ETR(II) during an induction curve measurement. Dark-adapted Col-0, not4a, Ws-4 and pgr3 plants were illuminated with 1000 μmol photons m−2 s−1 actinic light and a saturating flash was applied every 2 min to determine FM’ and PM’. The relative rates of electron transfer through PSI and PSII, ETR(I) and ETR (II), respectively, were calculated as in Fig. 2. Each point represents the average ±SD (n = 3–4).
