Fig. 1: Identification of NOT4-like proteins in Arabidopsis.

a Schematic diagram of protein domain structure and % amino acid identity for Sachharomyces cerevisiae (Sc) and Arabidopsis thaliana (At) NOT4 proteins. For full sequence alignments see Supplementary Fig. 1. b, c Representative rosette images of 4-week-old col-0, not4a-c mutants, and two independent not4a complementation lines (N4A-G1 and G3) grown under long day (LD) conditions. Bar = 1 cm. d Days to flowering and rosette leaf number at flowering for genotypes shown under short-day (SD) conditions (n = 10–12 per genotype). Box and whiskers plots show max and min, 25–75th percentiles (box), median (line) and mean (+). Letters indicate one-way ANOVA; Tukey’s test (p < 0.01). ns not significantly different. e Chlorophyll content (A, B and total) of SD grown rosette leaves from Col-0, not4a and two independent complementation lines (N4A-G1 and G3). Error bars = ±Standard Deviation. n = 5 biologically independent samples per genotype. Letters indicate one-way ANOVA; Tukey’s test (p < 0.05). f Lugol’s iodine staining of Col-0 and not4a rosette leaves. Bar = 1 cm. g Histochemical staining of 7-day-old seedlings of the N4A-G3 complementation line showing localisation of pNOT4::NOT4-GUS to the first true leaves. This correlates with where the pale-yellow phenotype first presents in not4a (arrowhead) relative to Col-0. Bar = 200 µm. Images are representative of >10 independent seedlings. h Detection of pNOT4::NOT4A-GUS by anti-GUS western blot in total vs chloroplast-specific protein extracts. LHCA4 is a chloroplast enriched protein control. UGPase is a cytosol control showing efficacy of chloroplast enrichment. RBCL was used as a loading control, and blots were repeated at least three times with similar results.