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 F_M’ and P_M’. 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.