Fig 2: NOT4A is required for normal photosynthetic function.a Volcano plot of up and down DEGs in not4a vs WT. The top 8 DEGs are labelled (UP: AT5G44925, QQS/AT3G30720 and DOWN: AT4G13575, AT4G09875, AT3G10430, AT4G13572, PILS7/AT5G65980, UNE15/AT4G13560). b Graphical representation of ‘cell part’ and ‘chloroplast’ Gene Ontology (GO) enrichment of DEGs in not4a vs WT. Asterisk refers to significant enrichment; Fisher’s exact test, p < 0.001. c Blue native (BN) gel of thylakoid protein complexes from Col-0, not4a and N4A-G3. Plants were grown under standard conditions (100 µmol photons m-2 s-1, 8 h/16 h light/dark), and the entire thylakoid network was solubilized with 1% (w/v) dodecyl maltoside (DM) and separated using BN gel electrophoresis. 50 µg of total protein was loaded. Red and blue arrows refer to PSII m/Cyt b6f and PSI-NDH mc bands, respectively, which are significantly depleted in not4a. See also Supplementary Fig. 4A. d Second dimension separation of DM-solubilised thylakoid proteins from Col-0 and not4a showing depletion of cytochrome b6f components PetA, PetB, PetC and PetD in not4a. The protein bands were identified based on Aro et al. 200536. e Western blot determination of relative protein abundance of NdhH and PetC subunits of the NDH and cytochrome b6f complexes in Col-0 and not4a, with coomassie blue (CBB) staining to show comparable protein loading. f Light-intensity dependence of ETR(I) and ETR(II). Dark-adapted Col-0 and not4a plants were subjected to illumination steps of 3 min at light intensities of 25–1000 µmol photons m-2 s-1 followed by a saturating flash (700 ms) to determine FM’ and PM’. The relative rates of electron transfer through PSI and PSII, ETR(I) and ETR (II), respectively, were calculated as Y(I) × PPFD × 0.84 × 0.5 and Y(II) × PPFD × 0.84 × 0.5. Each point represents the average ±SD (n = 5 biological replicates). Gels and blots in Fig. 2c–e were repeated at least three times with similar results.

Fig 3: Expression of protein complexes residing in thylakoid membranes during thylakoid biogenesis.Membrane protein fractions were isolated from Synechococcus cells grown under GL, HL, and HL-grown cells transferred to LL for 5 days. a Scheme of studied subunits from photosystem II (PSII), photosystem I (PSI), cytochrome b6f (Cyt b6f), type 1 NAD(P)H dehydrogenase-like complex (NDH-1), and ATPase. Illustration credit: Dr Tuomas Huokko, University of Liverpool; Relative protein quantification using label-free mass spectrometry data-dependent acquisition of subunits belonging to panel (b), PSI; (c), PSII; (d), Cyt b6f and (e), NDH-1. r.u., relative unit. Values are represented as means from three biologically independent experiments. All values are normalized to GL-samples. Transparent symbols represent three independent biological replicates. Fitted curves are fourth-degree polynomial functions. The corresponding adjusted R2 values for fitting are: PsaA, 0.98151; PsaB, 0.99498; PsaC, 0.99935; PsaL, 0.99967; PsbA, 0.99936; PsbD, 0.97432; PsbB, 0.96176; PsbC, 0.99982; PsbF, 0.99486; PsbL, 0.97164; PetA, 0.99612; PetB, 0.99497; PetC, 0.88053; PetD, 0.91018; NdhD4, 0.94908; NdhF4, 0.88885; NdhF3, 0.52203; CupA, 0.97552; NdhE, 0.61526. The equations of fitted curves are shown in Supplementary Table 1.

Fig 4: Content of key protein subunits in Synechococcus (A, C, E, G, I, K) and Synechocystis (B, D, F, H, J, L) over a 120 hour iron depletion time course.Data were compiled from 6 (Synechococcus) or 5 (Synechocystis) replicate immunoblots of cyanobacterial protein extracts from 6 (Synechococcus) or 5 (Synechocystis) replicate time course experiments (A, B) IsiA, (C, D) PsaC, (E, F) PetC,(G, H) PsbA, (I, J) PsbD, (K, L) AtpB. Protein subunit contents are expressed in femtomoles of protein per µg of total cellular protein. Data presented are mean +/- standard error, n = 5 or 6. Curve fits are second order polynomials with 95% confidence intervals plotted as outer dotted lines, except for Figure 2A which was fit with an logistic growth function, since IsiA reached a clear plateau.

Fig 5: Accumulation dynamics of photosynthesis-related proteins during de-etiolation.Three-day-old etiolated seedlings of Arabidopsis thaliana were illuminated for 0 hr (T0), 4 hr (T4), 8 hr (T8), 12 hr (T12), 24 hr (T24), 48 hr (T48), 72 hr (T72), and 96 hr (T96) under white light (40 µmol/m2/s). (A) Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane and immunodetected with antibodies against PsbA, PsbD, PsbO, PetC, PsaD, PsaC, Lhcb2, AtpC, ELIP, POR, phyA, HY5, and ACTIN proteins. (B–C) Quantification of PsbA, PetC, and PsaC during de-etiolation. Heatmap (B) was generated after normalization of the amount of each protein relative to the last time point (T96). Graph (C) corresponds to the absolute quantification of proteins at T96. Error bars indicate ± SD (n = 3). Quantification of photosystem-related proteins during de-etiolation is detailed in Figure 6—figure supplement 1.Figure 6—source data 1.Quantitative data for immunoblot analysis.