Figure legend supplementary figures
Supplementary figure S1. Nucleotide sequences (left) and amino acids sequences (right) of alb4 mutant lines #437 and #771. Full length alb4 was amplified from WT-ER, #437 and #771 and translated into the amino acid sequence (http://www.expasy.ch). (A) Nucleotide sequence of #437 with the point mutation indicted in red. Translated amino acid sequence with the premature stop codon in red. (B) Nucleotide sequence of the smaller amplification product of #771. Remaining sequence of exon 2 is labelled in red. Translated amino acid sequence of exon 1, eight amino acids of exon 2 and the beginning of exon 3 (bold black). (C) Nucleotide sequence of the larger amplification product of line #771. Sequence of intron 2 is labelled in red. Translated amino acid sequence showing the stop codons obtained through a frame shift.
Supplementary figure S2. Electron microscopy reveals alterations in the thylakoid structure of Arabidopsis alb4 loss-of-function mutants. (A) – (F) Electron micrographs of mesophyll cells of three-week-old wild-type and mutant plants. (A) – (C) show overviews of cells from wild-type (A) and the mutant lines #437 (B) and #771 (C) at a magnification of 1100x. Bar 10 µm. Panels (D) – (F) show chloroplast and thylakoid sections at magnifications of 12000x and 36000x from wild-type (D), line #437 (E) and line #771 (F). Bar, 500 nm.
Supplementary figure S3. Two-dimensional gel electrophoresis of chloroplast protein complexes on Blue-native PAGE and SDS-PAGE gels. Several independent samples of chloroplasts or isolated thylakoids were solubilized with 1% dodecylmaltoside from three-weeks-old wild-type and alb4 mutant plants and fractionated by BN-PAGE. An example of two independent experiments is given. (A) Examples of lanes from a native gel. The macromolecular protein complexes form thylakoid membranes are indicated at the top as follows: PSI-M, PSI monomer; PSII-SC, PSII supercomplexes; LHCI, light-harvesting complex of PSI; PSII-D, PSII dimer; LHCII, light-harvesting complex of PSII; LHCII-T, PSII light-harvesting complex trimer; LHCII-M, PSII light-harvesting complex monomer. (B) Lanes from BN-PAGE gels were horizontally laid on the top of an SDS-PAGE gel (15 % polyacriylamide, 4 M urea) followed by Coomassie staining. Differences between wild-type and alb4 mutants are indicated by circles. D, proteins present in decreased amounts in the mutant line relative to the wild-type. I, proteins present in increased amounts in the mutants. Sizes of protein markers are given in kDa on the left.
Supplementary figure S4. Transcription of the atp genes encoding ATP synthase subunits is not affected by depletion of Alb4. cDNA was synthesised form three-week-old wild-type and alb4 mutant lines and analysed by real-time PCR using appropriate primers. As an internal standard 18S rRNA was amplified. Each bar represents the mean (±SD) of at least three independent experiments. The genes atpA-E code for the five subunits of the hydrophilic CF1 portion of the ATP synthase complex, CF1a, CF1b, CF1γ, CF1δ and CF1e respectively; atpF-I encode the CF0I, CF0II, CF0 III and CF0IV subunits of the membrane-embedded CF0.
Supplementary figure S5. Alb4 and ATP synthase are localised in stroma-exposed thylakoids. Isolated thylakoids from three-week-old wild-type plants were solubilised with 0.1 % digitonin, and fractionated into appressed grana thylakoids (10K), margins (40K) and stroma-exposed lamellae (140K), as described by Ossenbühl et al. (2002). 10K, 40K and 140K designated membrane fractions pelleted by centrifugation at 10000xg, 40000xg and 140000xg respectively. Thylakoid fractions (equivalent to 5 µg of chlorophyll), and TCA-precipitated soluble proteins (20 µl) were fractionated by SDS-PAGE (13 % acrylamide, 4 M urea) and visualised either by Coomassie staining (A) or by immunodetection (B). (A) Thylakoid protein complexes from wild-type, stained with Coomassie, are designated as follows: core subunits of PSI; hydrophilic subunits α and b of ATP synthase; core subunits D1 and D2 of PSII; LHCII, light-harvesting complex II; PSI reaction centre subunit II; small subunits of PSI. (B) The gel blots were probed with antisera specific for Alb4, Alb3, ATP synthase subunits (CF0I and CF1α/b), PSI (psaF) as a marker for non-appressed membranes and PSII (core protein D1) as a marker protein for appressed membranes. T, solubilised thylakoids; SN, final supernatant. The sizes of the protein markers are given in kDa on the left.