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. 2012;7(12):e52100.
doi: 10.1371/journal.pone.0052100. Epub 2012 Dec 27.

Quality control of photosystem II: lipid peroxidation accelerates photoinhibition under excessive illumination

Tiffanie Chan  1 , Yurika Shimizu, Pavel Pospíšil, Nobuyoshi Nijo, Anna Fujiwara, Yoshito Taninaka, Tomomi Ishikawa, Haruka Hori, Daisuke Nanba, Aya Imai, Noriko Morita, Miho Yoshioka-Nishimura, Yohei Izumi, Yoko Yamamoto, Hideki Kobayashi, Naoki Mizusawa, Hajime Wada, Yasusi Yamamoto
Affiliations

Quality control of photosystem II: lipid peroxidation accelerates photoinhibition under excessive illumination

Tiffanie Chan et al. PLoS One. 2012.

Erratum in

  • PLoS One. 2013;8(5). doi:10.1371/annotation/8b0f8019-e4ba-4c35-9575-1a1313ae7b41

Abstract

Environmental stresses lower the efficiency of photosynthesis and sometimes cause irreversible damage to plant functions. When spinach thylakoids and Photosystem II membranes were illuminated with excessive visible light (100-1,000 µmol photons m(-1) s(-1)) for 10 min at either 20°C or 30°C, the optimum quantum yield of Photosystem II decreased as the light intensity and temperature increased. Reactive oxygen species and endogenous cationic radicals produced through a photochemical reaction at and/or near the reaction center have been implicated in the damage to the D1 protein. Here we present evidence that lipid peroxidation induced by the illumination is involved in the damage to the D1 protein and the subunits of the light-harvesting complex of Photosystem II. This is reasoned from the results that considerable lipid peroxidation occurred in the thylakoids in the light, and that lipoxygenase externally added in the dark induced inhibition of Photosystem II activity in the thylakoids, production of singlet oxygen, which was monitored by electron paramagnetic resonance spin trapping, and damage to the D1 protein, in parallel with lipid peroxidation. Modification of the subunits of the light-harvesting complex of Photosystem II by malondialdehyde as well as oxidation of the subunits was also observed. We suggest that mainly singlet oxygen formed through lipid peroxidation under light stress participates in damaging the Photosystem II subunits.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Light-induced decrease in the PS II activity of spinach thylakoids and PS II membranes monitored by chlorophyll fluorescence Fv/Fm.
(A) The thylakoids. (B) The PSII membranes. The samples were incubated in solution B and kept in the dark or illuminated with white light with given light intensities (100–1,000 µmol photons m−2 s−1) at either 20°C (brown bars) or 30°C (green bars) for 10 min. Dark control kept at 4°C is also shown (blue bars). After incubation in the dark for 30 min on ice, chlorophyll fluorescence was measured with a Mini-PAM at 25°C. The data are the means of three measurements±S.D.
Figure 2
Figure 2. Lipid peroxidation induced by illumination or by the addition of soybean lipoxygenase to the thylakoids and PS II membranes.
(A) Lipid peroxidation in the thylakoids (green bars) and PSII membranes (yellow bars) illuminated with high light (intensity, 1,000 µmol photons m−2 s−1) at 20°C for indicated periods. Lipid peroxidation was assayed by the TBARS assay. (B) Lipid peroxidation assayed using a fluorescence probe Spy-LHP in the thylakoids (green bars) and PSII membranes (yellow bars) illuminated either at 20°C or 30°C for 30 min with high light (the intensity was the same as that used in (A). Dark controls at 20°C and 30°C are also shown. (C) Lipid peroxidation in the thylakoids (green bars) and PSII membranes (yellow bars) induced by incubation with lipoxygenase (LOX, 0.1 mg mL−1) for indicated periods. The TBARS assay was used for lipid peroxidation assay. (D) Lipid peroxidation in the thylakoids (green bars) and PSII membranes (yellow bars) induced by incubation with LOX at either 20°C or 30°C for 30 min. The concentration of LOX was 0.1 mg mL−1. Spy-LHP was used for lipid peroxidation assay. The data are the means of three measurements±S. D.
Figure 3
Figure 3. Formation of malondialdehyde-protein adducts in the thylakoids under light stress or in the presence of lipoxygenase.
Spinach thylakoids were either illuminated with strong illumination (1,000 µmol photons m−2 s−1 for 30 and 60 min, denoted as “Light” at the top of the gel), or treated with LOX (0.1–0.5 mg mL−1, denoted as “+LOX”), and the production of malondialdehyde (MDA)–protein adducts was examined by Western blot analysis with an anti-MDA monoclonal antibody (Nichiyu, Japan). The molecular markers are shown at the left side of the fluorogram.
Figure 4
Figure 4. Effects of lipoxygenase on PSII activity in the thylakoids and PSII membranes.
To monitor the PSII activity, chlorophyll fluorescence Fv/Fm was measured. (A) The thylakoids. (B) The PSII membranes. LOX was added at the concentration of 0.1, 0.2 and 0.5 mg mL−1, and the reaction mixtures were incubated at either 20°C (brown bars) or 30°C (green bars) for 30 min. Controls incubated at 4°C without LOX are also shown (blue bars). The data are the means of three measurements±S. D.
Figure 5
Figure 5. Oxidation and subsequent aggregation and degradation of the LHCII and the D1 protein in the thylakoids by the addition of lipoxygenase (LOX).
(A) A fluorogram of Oxi-Blot analysis showing oxidation, aggregation and degradation of the proteins in the thylakoids by the addition of LOX. The concentration of LOX is 0.1, 0.2 and 0.5 mg mL−1. Other conditions were the same as those in Fig. 3. The position of Lhcb1 is indicated at the fluorogram on the right side. (B) Fluorograms showing aggregation of the D1 protein (left) and degradation of the D1 protein (right) by the addition of 0.5 mg mL−1 LOX. Western blot analysis was done using an antibody against the DE-loop of the D1 protein. Molecular markers and the positions of the D1 protein, D1 aggregates, and D1 degradation products are shown on the left side and right side of the fluorograms, respectively. The degradation products of the D1 protein by the addition of LOX was detected by overexposure of the gel (right).
Figure 6
Figure 6. Production of 1O2 in PSII membranes by strong illumination and addition of lipoxygenase.
(A) TEMPONE EPR spectra were measured in PSII membranes exposed to strong illumination (intensity, 100–1,000 µmol photons m−2 s−1, illumination time 30 min). The illumination was performed in the presence of 50 mM TMPD, 500 µg chlorophyll mL–1 and 40 mM MES (pH 6.5). (B) TEMPONE EPR spectra were measured in PSII membranes treated with LOX (0.1, 0.2 and 0.5 mg mL−1). The LOX treatment was performed in the presence of 50 mM TMPD, 500 µg chlorophyll mL–1 and 25 mM phosphate buffer (pH 7). (C) TEMPONE EPR spectra measured in pure TEMPONE dissolved in DMSO. (D) Relative intensity of TEMPONE EPR spectra obtained after weak light (200 µmol photons m−2 s−1), LOX (0.1 mg mL–1), 50 nM TEMPONE and strong light (1,000 µmol photons m−2 s−1), LOX (0.5 mg mL–1), 100 nM TEMPONE. Signal intensity was evaluated as a relative height of the central peak. The vertical bars represent 1,200 relative units. The data are the means of three measurements±S. D.
Figure 7
Figure 7. A schematic diagram of lipid peroxidation and a model showing a pathway of damage to the D1 protein and LHCII which is triggered by lipid peroxidation.
(A) A detailed mechanism for lipid peroxidation and the related processes. (B) A diagram showing proposed pathways for the light-induced damage to the D1 protein and LHCII. (1) a well- known route showing the light-induced production of 1O2 at the reaction center of PSII and damage to the D1 protein. (2) a new pathway of damaging the D1 protein through lipid peroxidation and the subsequent formation of 1O2 and MDA suggested in the present study. (3) lipoxygenase-induced lipid peroxidation and damage to the D1 protein.
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Grants and funding

This work was supported by Grants-in-aid for Scientific Research 20570039 from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Y. Y.) and by the grants no. ED0007/01/01 Centre of the Region Haná for Biotechnological and Agricultural Research and no. CZ.1.07/2.3.00/20.0057 Operational Programme Education for Competitiveness from the Ministry of Education Youth and Sports, Czech Republic (P. P.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.