10 research outputs found

    Light-Induced Oxidative Stress, <em>N</em>-Formylkynurenine, and Oxygenic Photosynthesis

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    <div><p>Light stress in plants results in damage to the water oxidizing reaction center, photosystem II (PSII). Redox signaling, through oxidative modification of amino acid side chains, has been proposed to participate in this process, but the oxidative signals have not yet been identified. Previously, we described an oxidative modification, <em>N</em>-formylkynurenine (NFK), of W365 in the CP43 subunit. The yield of this modification increases under light stress conditions, in parallel with the decrease in oxygen evolving activity. In this work, we show that this modification, NFK365-CP43, is present in thylakoid membranes and may be formed by reactive oxygen species produced at the Mn<sub>4</sub>CaO<sub>5</sub> cluster in the oxygen-evolving complex. NFK accumulation correlates with the extent of photoinhibition in PSII and thylakoid membranes. A modest increase in ionic strength inhibits NFK365-CP43 formation, and leads to accumulation of a new, light-induced NFK modification (NFK317) in the D1 polypeptide. Western analysis shows that D1 degradation and oligomerization occur under both sets of conditions. The NFK modifications in CP43 and D1 are found 17 and 14 Angstrom from the Mn<sub>4</sub>CaO<sub>5</sub> cluster, respectively. Based on these results, we propose that NFK is an oxidative modification that signals for damage and repair in PSII. The data suggest a two pathway model for light stress responses. These pathways involve differential, specific, oxidative modification of the CP43 or D1 polypeptides.</p> </div

    Predicted locations of NFK modifications, NFK365-CP43 and NFK317-D1, in the <i>T. vulcanus</i> PSII structure [<b>2</b>].

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    <p>The OEC is shown in black, grey, and red. P<sub>680</sub> and YZ (green spacefill) are shown above the OEC. The CP43 and D1 backbones are displayed in pink and green, respectively. The side chain of Trp-365 in CP43 is in red spacefill. The side chain of Trp-317 in D1 is in blue spacefill. MS/MS detected tryptic peptides corresponding to fraction A (red and yellow combined), B (blue), and C (red) are highlighted. The image was rendered with the Pymol Molecular Grapics System (<a href="http://www.pymol.org" target="_blank">www.pymol.org</a>).</p

    Optical absorption of NFK-containing PSII peptides (A) and the model compounds (B), tryptophan, NFK, and kynurenine.

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    <p>(A) shows absorption spectra of NFK-containing peptide fractions A–C. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042220#pone.0042220.s002" target="_blank">Table S1</a>, for average retention times from 350 nm chromatograms. Fraction A is displayed as a solid line, fraction B as a dashed line, and fraction C as a dotted line. In (B), absorption spectra of 40 µM tryptophan (solid line), 40 µM NFK (dotted line), and 40 µM kynurenine (dashed line) are shown in water. Absorption spectra in A were derived from the HPLC chromatogram and are on an arbitrary y-scale (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042220#s4" target="_blank">Materials and Methods</a>). The spectra in B were measured on a Hitachi spectrophotometer (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042220#s4" target="_blank">Materials and Methods</a>).</p

    Representative 350 nm HPLC chromatograms of oxygen-evolving PSII with and without 2 mM NaCl or TMA.

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    <p>In (A), (C), and (E), samples were incubated in the dark at room temperature for two hours (controls). In (B), (D), and (F), samples were illuminated with ∼7,000 µmol photons m<sup>−2</sup> s<sup>−1</sup> of white light for two hours at 25°C. In (C) and (D), 2 mM NaCl was added just prior to the dark or light incubation. In (E) and (F), 2 mM TMA was added just prior to the dark or light incubation. Fraction A is filled with horizontal stripes, fraction B has solid fill, and fraction C is filled with dots. The chromatograms are displaced on the y-axis for presentation purposes. The tick increments are 0.020 A.U. See Supporting Information for average retention times and summary of light-induced changes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042220#pone.0042220.s002" target="_blank">Table S1</a>). Fraction C corresponds to fraction 1 in ref <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042220#pone.0042220-Dreaden1" target="_blank">[19]</a>.</p

    Fraction B detection (top) and yield change in fractions A and C (bottom) following high light illumination.

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    <p>The yields were calculated from the average of three-six different experiments. Peaks in the 350 nm chromatogram were integrated, and the area was divided by the area of the 220 nm chromatogram to correct for changes in yield of tryptic peptides. The error bars are one standard deviation. See Supporting Information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042220#pone.0042220.s002" target="_blank">Table S1</a>) for summary of fold changes and average retention times.</p

    SDS-PAGE (A) and Western blot (B) using an antibody specific for the C-terminus of the D1 protein (Agrisera) (B).

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    <p>Control PSII membranes were maintained in the dark (lanes 2–5; lanes 10–13) or exposed to high light (∼7,000 µmol photon m<sup>−2</sup> s<sup>−1</sup>) for two hours at 25°C (lanes 6–9; lanes 14–17). Samples were either untreated (lanes 2, 6, 10, 14) or treated with 1 mM Na<sub>2</sub>EDTA (lanes 3, 7, 11 and 15), 2 mM NaCl (lanes 4, 8, 12, and 16), or 0.15 mM ZnCl<sub>2</sub> (lanes 5, 9, 13, and 17). Lane 1 displays the molecular weight markers. In both dark and light experiments, 24 µg chl was loaded per lane.</p

    Representative 350 nm HPLC chromatograms of tryptic peptides derived from oxygen-evolving PSII (A–B), OEC-removed (TW) PSII (C–D), and TM (E–F).

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    <p>In (A), (C), and (E), samples were incubated in the dark at room temperature for two hours (control). In (B), (D), and (F), samples were illuminated with ∼7,000 µmol photons m<sup>−2</sup> s<sup>−1</sup> of white light for two hours at 25°C. Fraction A is filled with horizontal stripes, and fraction C is filled with dots. The chromatograms are displaced on the y-axis for presentation purposes. The tick increments are 0.085 A.U. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042220#pone.0042220.s002" target="_blank">Table S1</a> for average retention times and summary of light-induced changes. Fraction C corresponds to fraction 1 in ref <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042220#pone.0042220-Dreaden1" target="_blank">[19]</a>.</p

    Steady state rates of oxygen evolution of PSII membranes (A) and TM (B) during high light illumination and in the dark.

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    <p>In A, PSII membranes were kept in the dark at 25°C for two hours (blue). PSII membranes were exposed to a white light intensity of 500 (red) and 7,000 (green) µmol photons m<sup>−2</sup> s<sup>−1</sup> for two hours at 25°C. In B, TM were kept in the dark (black and red) or exposed to a white light intensity of 7,000 µmol photons m<sup>−2</sup> s<sup>−1</sup> at chlorophyll concentrations of 1.0 (blue) or 0.1 mg/ml (green). Oxygen evolution was assayed every 30 minutes and normalized to time zero. The data shown are an average of three to six experiments. The error bars are plus and minus one standard deviation. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042220#s4" target="_blank">Materials and Methods</a>, Photoinhibition, for experimental conditions.</p

    MS/MS analysis of NFK modifications in fractions A–C.

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    <p>MS/MS analysis of NFK modifications in fractions A–C.</p

    Structures of tryptophan, NFK, kynurenine, and PSII.

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    <p>(A) shows the chemical structures of NFK (+32 <i>m/z</i>) and kynurenine (+4 <i>m/z</i>). (B) shows models of PSII (top) and TW (bottom) PSII. Tris-washing removes the extrinsic subunits and OEC, or Mn<sub>4</sub>CaO<sub>5</sub> cluster. The core subunits (CP43, D1, D2 and CP47) and electron transfer cofactors (tyrosine z (Y<sub>z</sub>), P<sub>680</sub>, pheophytin (Pheo), plastoquinone A (Q<sub>A</sub>), and plastoquinone B (Q<sub>B</sub>)) are labeled. The water-splitting reaction at the OEC is shown. The ROS species generated in PSII and TW PSII during photoinhibition are indicated. The subunit colors are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042220#pone-0042220-g008" target="_blank">Figure 8</a>. CP43 (pink); D1 (green); D2 (light blue); CP47 (dark blue); extrinsic subunits (violet, yellow, and orange).</p
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