PsaA | PSI-A core protein of photosystem I

O2 evolution and protein profile of photosynthetic apparatus in A. thaliana seedlings with generational exposure to Fe deficiency. (A) SHR-trap 1445 c0, pH 7.7 s1 pH 7.7 s2, pH 7.7 s1 c2 seedlings were grown for 11 days in control AIS medium and net O2 evolution (expressed as ?mol O2 evolved min-1 mg chlorophyll-1) was measured under illumination at either 100 or 800 ?E m-2 s-1. Values are mean ± SE of three biological replicas, each consisting of at least 15 seedlings. Letters represent statistical differences, according to Student’s t-test, with p < 0.05. (B) Western blot analysis with antibodies against PsaA, CP43, PsbO, D1, LHCII, and PsbE of thylakoid membranes purified from 11 days-old SHR-trap 1445 c0 seedlings germinated in -Fe AIS medium or from 11 days-old SHR-trap 1445 c0, pH 7.7 s1, pH 7.7 s1 pH 7.7 s2, pH 7.7 s1 c2 seedlings germinated in control (+Fe) AIS medium. Samples corresponding to 1 ?g chlorophyll were loaded on each lane.

Changes of PSII and PSI antenna and core protein levels. Proteins from control and Tl-treated white mustard leaves were separated by SDS-PAGE followed by immunodetection with antibodies against Lhcb1, Lhcb2, Lhca1 (antenna proteins) and D1, D2, CP43, PsbO, PsaA (core proteins). Samples were loaded on the equal amount of chlorophyll (0.25 ?g). Description of samples abbreviation as given in the legend to Fig. 3

Immunoblot analysis of photosynthetic complex accumulation in wild-type tobacco and the two ?psaI lines grown under low, intermediate, and high-light conditions. Because the accumulation of most tested proteins was highest under high-light conditions, lanes one to three contain samples diluted to 25%, 50%, and a 100% sample of wild-type tobacco grown under high-light conditions, to allow for semi-quantitative determination of changes in protein abundance. Lanes four and five contain the two transplastomic lines grown at 1000 µE m?2 s?1. Lanes six to eight contain wild-type tobacco and the mutants grown at intermediate light intensities, and lanes nine to eleven contain samples grown at low light intensities. For PSII, the accumulation of the essential subunits PsbB (CP43) and PsbD (D2) and the LHCB1 antenna protein were determined, while for the cytochrome b6f complex, the accumulation of the essential redox-active subunits PetA (cytochrome f), PetB (cytochrome b6), and PETC (Rieske FeS protein) was tested. AtpB was probed as an essential subunit of the chloroplast ATP. For PSI, in addition to the three essential plastome-encoded subunits PsaA, PsaB, and PsaC, the accumulation of the nuclear-encoded subunits PSAD, PSAH, PSAK, PSAL, and PSAN and of the four LHCI proteins (LHCA1, LHCA2, LHCA3, LHCA4) was determined. Finally, we examined the accumulation of Ycf4, the chloroplast-encoded PSI-biogenesis factor encoded in the same operon as PsaI, and the nuclear-encoded assembly factor Y3IP1.

Accumulation of photosynthetic complexes in the mutant strains.Cells were grown in TAP medium at 25?°C under LED white light (8??mol photons m?2?s?1) and collected at the mid-log phase. Two independent lines are shown for each construct. Protein samples were loaded on an equal chlorophyll basis (0.5??g per lane), and a dilution series of WT samples is shown for semi-quantitative comparison. Antibodies against essential subunits of PSII (D2), PSI (PsaA), cytochrome b6f (PetA) and ATP synthase (AtpB) probed the accumulation of the respective photosynthetic complexes. Numbers on the left side of the blots are molecular weights in kD. See Supplementary Fig. 9 for the uncropped blot images.

Immunolocalization of photosystems and of cyt b6f in the thylakoid membranes of P. tricornutum.(a–c) TEM images of P. tricornutum labelled with antibodies directed against the PsaA subunit of PSI (a), the PsbA subunit of PSII (b) and the PetA subunit of cyt b6f (c). (d) TEM micrograph of P. tricornutum thylakoid membranes showing four distinct areas: the internal membranes (‘core’: violet); the external, peripheral membranes (‘per.’: green); the pyrenoid (‘pyr.’: orange) and the envelope (‘env.’: magenta). Bars: 200?nm. (e) Principal component analysis of PSI, cyt b6f and PSII immunolocalization with the PsbA (solid squares), PsbC (open squares), PetA (cyan circle), PsaC (solid triangles) and PsaA (open triangles) antibodies. See also Supplementary Fig. 4. A total of 258 images from four independent cultures were analysed. The first two components represent more than 91% of the variance (see Supplementary Table 1, and Methods for a more detailed explanation). Green arrow: peripheral variable; violet arrow: core variable; orange arrow: pyrenoid variable; Magenta arrow: envelope variable. (f) 2D representation of the barycentre for the PSI (? PsaA+? PsaC antibodies, black square), cyt b6f (PetA, cyan circle) and PSII (? PsbA+? PsbC antibodies, red triangle) distributions. The point size along an axis is proportional to the s.d. along the corresponding component. (g) Solubilization of P. tricornutum thylakoid membranes with increasing concentrations of digitonin (0.1%, 0.2%, 0.5%, 1%). Pellet (P) and supernatant (S) were analysed by western blotting with the same anti PSI, PSII and cyt b6f antibodies as in a–c. Representative data set of an experiment replicated on three different biological samples.

Subcellular distribution of CrPCYA1 in chloroplast and the export of phycocyanobilin to cytosol. (A) Biochemical fractionation of chloroplast components. Antibodies against compartmental marker proteins distributed in different chloroplast fractions were employed to distinguish the presence of CrPCYA1 via immunoblot analysis of total proteins (T), soluble fraction (S), envelope membrane (E) and thylakoid membrane (TM) from isolated chloroplast. Tic40, chloroplast envelope marker; PsaA, thylakoid membrane marker. (B) Zinc-dependent fluorescence assay. Photosensory core module (PCM) region of the prasinophyte algal DtenPHY1 purified from Chlamydomonas CC400 covalently binds phycocyanobilin (PCB). Upper panel, zinc blot; lower panel, Coomassie blue stain (CB). The two-fold serial dilutions of E. coli LMG194/pPL-PCB expressed and purified DtenPHY1 were used as positive controls. –PCB, without addition of PCB; +PCB, assembly with PCB in vitro.

Detection of CP43 and PsaA proteins on a film (a) and quantification of these proteins (b) from control (white bars) and EL (black bars) cells. Western blotting was done from extracted total soluble proteins, and 1 µg (CP43) or 2 µg (PsaA) of total proteins were loaded to SDS-PAGE per well. Binding of the primary and secondary antibodies was visualized via luminescence emitted by alkaline phosphatase. The signals were normalized to the average of signals originating from control samples of the respective western blot. Each bar represents an average of three biological replicates and the error bars show SD

Analysis of PSI, PSII and LHCII content by immunoblots, P700 activity and functional PSII antenna size. (a, b) Immunoblot analysis of PSI (??PsaA antibody), PSII (??CP43 antibody) and LHCII (??LHCII antibody). Loading was performed on a chlorophyll basis: total ?g of chlorophylls loaded in each lane is reported on the top of Panel a and b. (c, d) PSI/PSII (c) and LHCII/PSII (d) ratios calculated by densitometry of immunoblot signals for C. sorokiniana (panel a, grey colour) and C. vulgaris (panel b, red colour) in AIR (full colour) or CO2 (dash colour) condition. (e) Maximal P700 oxidation on a chlorophyll basis in C. sorokiniana (left, grey colour) and C. vulgaris (right, red colour) in AIR (full colour) or CO2 (dash colour) normalized to AIR condition. (f) Functional antenna size of the photosystem II (1/?2/3) normalized to AIR condition in C. sorokiniana (grey colour) and C. vulgaris (red colour). Data are means of three biological replicates with standard deviation shown. Significant different values in CO2 versus AIR are indicated by ** (p?