Volume 2, Issue 3, Pages 416-429 (May 2009) The Role of Phosphorylation in Redox Regulation of Photosynthesis Genes psaA and psbA during Photosynthetic Acclimation of Mustard  Steiner Sebastian , Dietzel Lars , Schröter Yvonne , Fey Vidal , Wagner Raik , Pfannschmidt Thomas   Molecular Plant  Volume 2, Issue 3, Pages 416-429 (May 2009) DOI: 10.1093/mp/ssp007 Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 1 Video Imaging of Chl Fluorescence Parameter Fs/Fm in Mustard Seedlings Acclimated to PSI- or PSII-Light. (A) Picture under white light of PSI, PSI-II, PSII, and PSII-I seedlings placed in one rack for simultaneous Chl fluorescence detection. (B) Fs/Fm parameter of the same seedlings as in (A) shown in false colors. Red, high Fs/Fm; blue, low Fs/Fm. For integrated data of this picture, see Table 1. Molecular Plant 2009 2, 416-429DOI: (10.1093/mp/ssp007) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 2 Purification and Electrophoretic Separation of Chloroplast Protein Fractions with RNA Polymerase Activity. (A) Scheme of isolation procedure for chloroplast protein fractions with transcription activity. (B) Peak fractions after heparin-Sepharose chromatography and high salt step elution. Equal protein amounts (15 μg) of the peak fractions were separated on a 6%–16% SDS gel and stained with silver. Sizes of marker proteins separated in parallel are given in the left margin; subunits of E. coli RNA polymerase (E.c.) are shown as additional control. Molecular Plant 2009 2, 416-429DOI: (10.1093/mp/ssp007) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 3 Redox Control at the psaA Promoter and Promoter Structures of Plastid Genes psaA and psbA. (A) Primer extension assays at the psaA promoter. Plants were grown under PSI, PSI-II, PSII, and PSII-I growth light regimes and total RNA was isolated from each condition. A fluorescence dye-labeled primer was designed to anneal within the first 50 bp of the coding region of the psaA operon and was used both in a reverse transcription reaction with the isolated RNA and a sequencing reaction of chloroplast DNA fragments covering the psaA promoter region. Products were separated in parallel on a denaturing 4% acrylamide gel containing 7 M urea and detected by laser excitation in a Licor 4200 sequencer. A sequencer image of the primer extension analysis is shown. The DNA sequence within the psaA promoter is shown on the left, primer extension products on the right flanked by a magnification of the primer extension products. The detected 5′-end is marked by a dotted line; the respective transcription start nucleotide is given in bold letters. (B) Schematic presentation of promoter structures of plastid genes psaA and psbA. Transcription start site is given as +1 and marked with the same black dot as in Figure 3A. All other positions are given relative to it. White boxes indicate –35/–10 boxes, gray-shaded boxes the position of the psaA region U (left of transcription start site) and region D element (right of transcription start site), black boxes a TATA-like element. Black bars indicate the position of probes used for EMSAs or Southwestern experiments (SW). Molecular Plant 2009 2, 416-429DOI: (10.1093/mp/ssp007) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 4 DNA-Binding Protein Complexes in HS Fractions. A gray-scaled fluorescence image of the EMSA gel is shown. PSI-II or PSII-I heparin-Sepharose fractions were incubated with fluorescence-labeled core promoter probes for psaA and psbA (Figure 3B). After electrophoretic separation on a 6% native polyacrylamide gel, DNA–protein complexes were directly visualized using a fluorescence scanner. In order to test binding specificity, unspecific competitor (comp.) was added. The first two lanes represent negative probe controls (first lane: psaA; second lane: psbA). Signal 1 (PEP): binding signal of the plastid encoded RNA polymerase. Signals 2–8: seven different DNA-protein complexes. Signal 9: unbound, free DNA. Saturated signals in this area appear white/gray. Molecular Plant 2009 2, 416-429DOI: (10.1093/mp/ssp007) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 5 Southwestern Analysis of HS fractions. 30 μg of purified PSI-II and PSII-I fractions (I-II and II-I) were separated on a 6–16% SDS–polyacrylamide gradient gel, electro-blotted on a nitrocellulose membrane, re-natured and incubated with fluorescent dye-labeled psaA or psbA promoter probes. Signals indicate proteins able to bind the respective promoter fragment. Sizes of marker proteins separated in parallel are given in the left margin. The upper part of the psbA blot (indicated by a black line) is displayed with a higher contrast to visualize weaker signals. Molecular Plant 2009 2, 416-429DOI: (10.1093/mp/ssp007) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 6 Long-Term Protein Phosphorylation State of HS Fractions. Phosphorylation states were characterized by Western-immuno-detection after SDS–PAGE using anti-phospho-aminoacid antibodies directed against phospho-threonine (P-Thr), phospho-serine (P-Ser), and phospho-tyrosine (P-Tyr). Fluorescence-labeled secondary antibodies were detected using a fluorescence scanner. Black arrows mark proteins that exhibit a stronger signal in that sample to which the arrow is placed. Sizes of marker proteins separated in parallel are given in the left margin. Molecular Plant 2009 2, 416-429DOI: (10.1093/mp/ssp007) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 7 Short-Term Effect on Protein Phosphorylation State of HS Fractions. HS fractions were isolated from plants harvested just 1 h after the light shift. Phosphorylation states were characterized by Western-immuno-detection after SDS–PAGE using anti-phospho-aminoacid antibodies directed against phospho-threonine (P-Thr), phospho-serine (P-Ser), and phospho-tyrosine (P-Tyr). Fluorescence-labeled secondary antibodies were detected using a fluorescence scanner. Black dots mark phosphorylation signals that were not detected after the long-term treatment; white squares indicate signals that were detected after the long-term shift but lacking after the 1-h treatment. Sizes of marker proteins separated in parallel are given in the left margin. Molecular Plant 2009 2, 416-429DOI: (10.1093/mp/ssp007) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 8 Characterization of Endogenous Kinase Activity in the Heparin-Sepharose Fractions. Kinase activity from heparin-Sepharose purified proteins from PSI-II and PSII-I chloroplasts was assayed in in-vitro phosphorylation reactions using γ-32P-ATP. Radioactively labeled proteins were separated by SDS–PAGE and detected by a phosphorimager. Lanes 1 and 2: autophosphorylation of peak fractions after heparin-Sepharose chromatography. Radioactive bands are marked by arrows in the left margin and the respective apparent molecular weight is given. Lanes 3 and 4: silver-stained proteins in the dried gel used for autoradiography shown in lanes 1 and 2 are presented as loading control. Sizes of marker proteins separated in parallel are given in the right margin. Lanes 5 and 6: peak fractions were heat-inactivated and subjected to phosphorylation by an exogenously added PKA. Lanes 7 and 8: peak fractions were dephosphorylated using shrimp alkaline phosphatase, heat-inactivated and finally radioactively labeled by PKA. The strong additional band at 58 kDa represents the deactivated phosphatase. Lane 9: PKA autophosphorylation control. Molecular Plant 2009 2, 416-429DOI: (10.1093/mp/ssp007) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 9 Chloroplast Run-On Transcription Assays. Chloroplasts were isolated from PSI-II and PSII-I plants and used in either standard run-on transcription assays (left panels) or pre-incubation assays with H7 and DTT (right panels, only from PS-II plants). Transcriptional activity of psaA and psbA was determined with a phosphorimager and normalized to 18S rRNA. Activities of psaA and psbA in the left panels can be directly compared. Variations were between 5 and 10%. In pre-incubation assays, plastids from PSI-II plants were incubated for 20 min in the presence of PSII-light and indicated agents. Activities were determined and normalized to the control (–DTT, –H7) which is set as 100%. Two independent experiments were done in triplicate. Standard deviation is given. Molecular Plant 2009 2, 416-429DOI: (10.1093/mp/ssp007) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions