PsbA | D1 protein of PSII, C-terminal (rabbit antibody) (thylakoid membrane marker)

AtTSPO accumulation and chloroplast localization upon salt stress. (A) Immunoblot analysis of OxAtTSPO:eGFP (OxM1TSPO:eGFP, OxM21TSPO:eGFP and OxM42TSPO:eGFP) fusion proteins detected in plants with an antibody to GFP. Plants were untreated, or treated with 150 mM NaCl. As a control wild-type plants and plants over-expressing GFP (OxeGFP) seedlings were used. (B) Anti-GFP immunoblot of trypsinized chloroplasts from Arabidopsis plants either untreated or treated with 150 mM NaCl. Control immunoblots were probed with antibodies chloroplast proteins RuBisCo and D1; and to cytosolic UGPase. Each lane represents equal amounts of chloroplasts.

Membrane protein complexes of PSII and PSI survive in the 35S:A9 seedlings.(A) Western detection of complexes after the H2O2 treatments. The PSII complexes (top) separated by BN-PAGE were detected using anti-D1 (DE-loop) antibodies at 1/5,000 dilution. The PSI complexes (bottom) were detected using anti-PsaB antibodies at 1/4,000 dilution. PSII symbols: •, and the bracket on top respectively mark the dimeric PSII complex, and the PSII-LHCII super-complexes. The asterisk marks the CVII (CP43-less PSII monomer) complex. PSI symbols: • marks the PSI-LHCI super-complex that co-migrates in our gel system with the dimeric PSII complex; the asterisk marks the PSI monomer. (B) The PSII complexes also withstand drastic dehydration. The thylakoid samples were analyzed immediately (0 h) after the dehydration treatment (DT), and following rehydration for 16 h, DT (16 h). In this case the complexes were detected using anti-D1 (C-terminal) antibodies at 1/15,000 dilution. An additional PSII complex mentioned in the text is indicated: CV (**, PSII monomer).

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.

Thylakoid protein phosphorylation and state transitions are disturbed after high light treatment in pgr6 background. a Total protein extracts of 4-week-old wild type (WT), pgr6-1, pgr6?2, sps2 and stn7/stn8 analysed by immunoblotting with anti-phosphothreonine antibody; the principal thylakoid phospho proteins are indicated on the right according to their size. Core photosystem II proteins D1 (PsbA) and D2 (PsbD) are indicated as a single band due to their poor resolution. Actin was used as a loading control. b Lhcb1 and Lhcb2 phosphorylation levels were visualised after separation on Phostag™-pendant acrylamide gels. The upper band corresponds to the phosphorylated form (p-), stn7/stn8 double mutant is a non-phosphorylated control. c Average transient of the variable room temperature chlorophyll fluorescence measured during the transition from red (660nm) supplemented with far-red light (720nm) state 1 to pure red light state 2 (n?=?4 independent pots containing 2–3 plants). The fluorescence curves from pgr6 and sps2 are shifted on the x-axis to allow visualising the FMST1 and FMST2 values. The x-axis time scale refers to the wild-type curve. d Calculated quenching related to state transition (qT?=?(FMST1?–?FMST2)/FM), expressed as the percentage of FM that is dissipated by the state 1 to state 2 transition, of wild type (WT), pgr6?1 and sps2 under moderate light (120?mol?m?2?s?1) (ML) and after 3?h of high light (500?mol?m?2?s?1) (HL). Whiskers and box plot shows the minimum, first quartile, median, average, third quartile and maximum of each dataset (n?=?4 biologically independent samples); p-values are calculated via a two-tailed Student’s t test. e STN7 phosphorylation level visualised after separation on Phostag™-pendant acrylamide gels. The upper band corresponds to the phosphorylated form (p-), a protein sample from stn7/stn8 double mutant was loaded as a control for the antibody specificity. Uncropped images of the membranes displayed in a, b and e are available as Supplementary Fig. 11. Data points for items c, d are available as Supplementary data 2

High light?induced changes of the D1 and ZEP protein content. Detached leaves of Arabidopsis (a), pea (b), spinach (c) and tobacco (d) plants were vacuum?infiltrated with H2O or 3 mM SM, as indicated in each panel. Leaves were exposed to HL for 8 hr at 2,000 µmol photons m?2 s?1 and 4°C and then transferred to LL (10–20 µmol photons m?2 s?1) for 16 hr. Total leaf protein extracts equivalent to 5 µg protein were separated by SDS?PAGE. The abundance of ZEP and D1 protein, as well as of the large subunit of RubisCO (RbcL) was assessed by immunoblotting with specific antibodies. Representative blots from at least 3 biological replicates are shown

High light?induced changes of the D1 and ZEP protein content. Detached leaves of Arabidopsis (a), pea (b), spinach (c) and tobacco (d) plants were vacuum?infiltrated with H2O or 3 mM SM, as indicated in each panel. Leaves were exposed to HL for 8 hr at 2,000 µmol photons m?2 s?1 and 4°C and then transferred to LL (10–20 µmol photons m?2 s?1) for 16 hr. Total leaf protein extracts equivalent to 5 µg protein were separated by SDS?PAGE. The abundance of ZEP and D1 protein, as well as of the large subunit of RubisCO (RbcL) was assessed by immunoblotting with specific antibodies. Representative blots from at least 3 biological replicates are shown

High light?induced changes of the D1 and ZEP protein content. Detached leaves of Arabidopsis (a), pea (b), spinach (c) and tobacco (d) plants were vacuum?infiltrated with H2O or 3 mM SM, as indicated in each panel. Leaves were exposed to HL for 8 hr at 2,000 µmol photons m?2 s?1 and 4°C and then transferred to LL (10–20 µmol photons m?2 s?1) for 16 hr. Total leaf protein extracts equivalent to 5 µg protein were separated by SDS?PAGE. The abundance of ZEP and D1 protein, as well as of the large subunit of RubisCO (RbcL) was assessed by immunoblotting with specific antibodies. Representative blots from at least 3 biological replicates are shown

High light?induced changes of the D1 and ZEP protein content. Detached leaves of Arabidopsis (a), pea (b), spinach (c) and tobacco (d) plants were vacuum?infiltrated with H2O or 3 mM SM, as indicated in each panel. Leaves were exposed to HL for 8 hr at 2,000 µmol photons m?2 s?1 and 4°C and then transferred to LL (10–20 µmol photons m?2 s?1) for 16 hr. Total leaf protein extracts equivalent to 5 µg protein were separated by SDS?PAGE. The abundance of ZEP and D1 protein, as well as of the large subunit of RubisCO (RbcL) was assessed by immunoblotting with specific antibodies. Representative blots from at least 3 biological replicates are shown

Double mutant maintains thylakoid protein phosphorylation and state transitions after high light. (A) Total protein extracts of wild type (WT), abc1k1.1, -2, abc1k3.1, -2, and abc1k1/abc1k3.1, -2 light-exposed leaves were separated by SDS PAGE, transferred on nitrocellulose membrane and decorated with anti-phosphothreonine antibody. The main thylakoid phospho-proteins are indicated on the right according to their size. Core photosystem II proteins D1 (PsbA) and D2 (PsbD) are indicated together due their poor resolution. (B) The accumulation of the principal photosynthetic complexes was assessed using antibodies against specific subunits of each complex: anti-Lhcb2 for the major LHCII, anti-D1 (PsbA) for PSII, anti-PetC for cytochrome b6f, anti-PsaD and anti-PsaC for PSI, and anti-AtpC for ATP synthase. Actin signal is shown as a loading control. (C) Fluorescence quenching related to the state transitions (qT) of wild type (WT), abc1k1.1, -2, abc1k3.1, -2, and abc1k1/abc1k3.1, -2 under moderate light (120 ?mol of photons m–2 s–1) (ML) and after 3 h of high light (500 ?mol of photons m–2 s–1) (HL). qT was calculated from the maximal chlorophyll fluorescence measured after 10 min exposure to red light (660 nm) supplemented with far-red illumination (720 nm) “State 1” (FMST1) or to pure red light “State 2” (FMST2). Quenching related to state transition was calculated as qT = (FMST1 – FMST2)/FM. Each value represents the average of a pot containing 2–3 plants. Superscript letters are used to indicate statistically different groups (p < 0.05) by paired Student’s t-test.