Production of superoxide from Photosystem II in a rice (Oryza sativa L.) mutant lacking PsbS

Background PsbS is a 22-kDa Photosystem (PS) II protein involved in non-photochemical quenching (NPQ) of chlorophyll fluorescence. Rice (Oryza sativa L.) has two PsbS genes, PsbS1 and PsbS2. However, only inactivation of PsbS1, through a knockout (PsbS1-KO) or in RNAi transgenic plants, results in plants deficient in qE, the energy-dependent component of NPQ. Results In studies presented here, under fluctuating high light, growth of young seedlings lacking PsbS is retarded, and PSII in detached leaves of the mutants is more sensitive to photoinhibitory illumination compared with the wild type. Using both histochemical and fluorescent probes, we determined the levels of reactive oxygen species, including singlet oxygen, superoxide, and hydrogen peroxide, in leaves and thylakoids. The PsbS-deficient plants generated more superoxide and hydrogen peroxide in their chloroplasts. PSII complexes isolated from them produced more superoxide compared with the wild type, and PSII-driven superoxide production was higher in the mutants. However, we could not observe such differences either in isolated PSI complexes or through PSI-driven electron transport. Time-course experiments using isolated thylakoids showed that superoxide production was the initial event, and that production of hydrogen peroxide proceeded from that. Conclusion These results indicate that at least some of the photoprotection provided by PsbS and qE is mediated by preventing production of superoxide released from PSII under conditions of excess excitation energy. Electronic supplementary material The online version of this article (doi:10.1186/s12870-014-0242-2) contains supplementary material, which is available to authorized users.

. Light response curves for NPQ.
3 Figure S3. Light response curves for electron transport rates.
Plants were grown on solid (agar) Murashige and Skoog media under different light conditions; a low-light (LL), 30 µmol photons m -2 s -1 ; a medium-light (ML), 300 µmol photons m -2 s -1 ; a natural fluctuating light in a greenhouse (FHL), 50 to 1,500 µmol photons m -2 s -1 . All plants were grown on agar plate at the same growth conditions in the culture room except the light intensity; for FHL which grown in greenhouse under fluctuating light. To measure growth rate we have used plants heights only. Each point represents mean of at least 4 experiments (SD indicated by bar) and the asterisks denote the results that were significantly different from those in the wild type (*P < 0.05).
The statistical significance was evaluated using the t-test.
Root systems of 1-week-old seedlings were infiltrated with DanePy (2 mM) for 1 h under darkness.
Merged confocal fluorescence images of leaves from wild type (WT) and PsbS-KO taken before (dark) and after (light) exposure to photoinhibitory illumination (1,000 µmol photons m -2 s -1 To detect singlet oxygen in samples of our wild type and mutant plants we used the fluorescent sensor DanePy (Hideg et al., 2002), synthesized as described by Kàlai et al. (2002). We tested singlet oxygen production in isolated thylakoids to check the quality of our synthesized DanePy; singlet oxygen production in isolated thylakoids was carried out as described in Fufezan et al. (2002) in the presence of two types of herbicide (DCMU and bromoxynil) (data not shown). The fluorescence from DanePy and Chl was then monitored using a Zeiss (Oberkochen, Germany) LSM510 confocal laser scanning microscope with Kr/Ar laser excitation. Leaves of one-week-old rice seedlings were imaged after infiltration with DanePy with a 25X objective by using 364 nm laser excitation. Channel mode detection for 10 min at room temperature). Distribution plot for intensities of all 1024 x 1024 pixels is shown beneath each digitized confocal image. Similar results were obtained from 6 independent experiments. was used to record the emissions of DanePy (505 to 550 nm). A single channel was used throughout an entire experiment and corrected for autofluorescence and Chl fluorescence by collecting a 488-nm excitable signal before photoinhibitory treatment.
Image acquisition conditions were kept constant for comparison between control and illuminated leaves. Confocal images were analyzed using LSM 510 Image Examiner software. Whole confocal images were used to make the pixel distribution plots, and the distribution of dark pixels was ignored. The selected experimental conditions, including excitation UV-laser beam intensity and emission detection threshold, were as described by Hideg et al. (2006) to minimize autofluorescence from the leaves. 7 8 Figure S6. Detection of superoxide anion radical production.
Leaves of wild-type (WT) and PsbS-KO seedlings were infiltrated with dihydroethidium (25 µM) under darkness. Merged confocal fluorescence images were taken before (dark) and after (light) exposure to photoinhibitory illumination (1,000 µmol photons m -2 s -1 for 10 min at room temperature). Distribution plot for intensities of all 1024 x 1024 pixels is shown below each digitized image. Similar results were obtained from 6 independent experiments. Pixel distribution plots showed that after 10 min of photoinhibitory illumination in both wild type and PsbS-KO rice leaves, the frequencies of the low intensity pixels decreased with subsequent increases in the frequencies of the high intensity pixels, and this shift was more apparent in PsbS-KO rice leaves.
Similar results were obtained in six independent experiments. Channel mode detection was used to record the emissions of DHE (550 to 650 nm). Another experimental conditions are the same as in Figure S5 legend. 9 Figure S7. Detection of hydrogen peroxide production.
Leaves from wild-type (WT) and PsbS-KO seedlings were infiltrated with 2',7'-dichlorofluorescein diacetate (DCFDA) (10 µM) under darkness. Merged confocal fluorescence images were taken before (dark) and after (light) exposure to photoinhibitory illumination (1,000 µmol photons m -2 s -1 for 10 min at room temperature). Distribution plot for intensities of all 1024 x 1024 pixels is shown beneath each digitized image. Similar results were obtained from 6 independent experiments. Pixel distribution plots indicated that DCFDA fluorescence increased more in PsbS-KO leaves than in wild-type leaves after photoinhibitory illumination for 10 min. Similar results were obtained in six independent experiments. Channel mode detection was used to record the emissions of DCFDA (505 to 550 nm). Another experimental conditions are the same as in Figure S5 legend.  Redox difference spectra measured in Mn-depleted (Tris-treated) PSII complexes under darkness and after illumination. Samples (100 μg Chl mL -1 ) were illuminated with white light (1,000 μmol photons m −2 s −1 ). Dark lines − hydroquinone minus ferricyanide spectra; Blue lines -100-s illumination minus ferricyanide-oxidized spectra; Red lines -300-s illumination minus ferricyanideoxidized spectra.
14 Figure S11. Protein composition of thylakoids and BBY particles.
The thylakoids and BBY particles of the wild-type and PsbS-KO mutant plants were subjected to SDS-PAGE and stained with coomassie. M, molecular weight markers; WT, wild type; PsbS, PsbS-