Photosystem II (PSII) is an intrinsic membrane protein complex that functions as a light-driven water:plastoquinone oxidoreductase in oxygenic photosynthesis. Electron transport in PSII is associated with formation of reactive oxygen species (ROS) responsible for oxidative modifications of PSII proteins. In this study, oxidative modifications of the D1 and D2 proteins by the superoxide anion (O2•−) and the hydroxyl (HO•) radicals were studied in WT and a tocopherol cyclase (vte1) mutant, which is deficient in the lipid-soluble antioxidant α-tocopherol. In the absence of this antioxidant, high-resolution tandem mass spectrometry was used to identify oxidation of D1:130E to hydroxyglutamic acid by O2•− at the PheoD1 site. Additionally, D1:246Y was modified to either tyrosine hydroperoxide or dihydroxyphenylalanine by O2•− and HO•, respectively, in the vicinity of the nonheme iron. We propose that α-tocopherol is localized near PheoD1 and the nonheme iron, with its chromanol head exposed to the lipid–water interface. This helps to prevent oxidative modification of the amino acid’s hydrogen that is bonded to PheoD1 and the nonheme iron (via bicarbonate), and thus protects electron transport in PSII from ROS damage.

Hydrogen peroxide (H2O2) is known to be generated in Photosystem II (PSII) via enzymatic and non-enzymatic pathways. Detection of H2O2 by different spectroscopic techniques has been explored, however its sensitive detection has always been a challenge in photosynthetic research. During the recent past, fluorescence probes such as Amplex Red (AR) has been used but is known to either lack specificity or limitation with respect to the minimum detection limit of H2O2. We have employed an electrochemical biosensor for real time monitoring of H2O2 generation at the level of sub-cellular organelles. The electrochemical biosensor comprises of counter electrode and working electrodes. The counter electrode is a platinum plate, while the working electrode is a mediator based catalytic amperometric biosensor device developed by the coating of a carbon electrode with osmium-horseradish peroxidase which acts as H2O2 detection sensor. In the current study, generation and kinetic behavior of H2O2 in PSII membranes have been studied under light illumination. Electrochemical detection of H2O2 using the catalytic amperometric biosensor device is claimed to serve as a promising technique for detection of H2O2 in photosynthetic cells and subcellular structures including PSII or thylakoid membranes. It can also provide a precise information on qualitative determination of H2O2 and thus can be widely used in photosynthetic research.

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This article contains data related to the research article entitled, “Organic radical imaging in plants: Focus on protein radicals” (Kumar et al., 2018). The data presented herein focus on reactive oxygen species (ROS) and organic radical formed within photosynthetic tissues of Arabidopsis thaliana during high light stress and includes (1) Confocal laser scanning microscopic images using 3′-p-(hydroxyphenyl) fluorescein (HPF) as specific probe for the detection of hydroxyl radical (HO•); (2) Confocal laser scanning microscopic images using Singlet Oxygen Sensor Green (SOSG) as a specific probe for the detection of singlet oxygen (1O2) and; (3) Electron paramagnetic resonance (EPR) spectroscopy using spin traps for the detection of organic radical.

The light-driven splitting of water to oxygen (O 2 ) is catalyzed by a protein-bound tetra-manganese penta-oxygen calcium (Mn 4 O 5 Ca) cluster in Photosystem II. In the current study, we used a large-scale integration (LSI)-based amperometric sensor array system, designated Bio-LSI, to perform two-dimensional imaging of light-induced O 2 evolution from spinach leaves. The employed Bio-LSI chip consists of 400 sensor electrodes with a pitch of 250 μm for fast electrochemical imaging. Spinach leaves were illuminated to varying intensities of white light (400–700 nm) which induced oxygen evolution and subsequent electrochemical images were collected using the Bio-LSI chip. Bio-LSI images clearly showed the dose-dependent effects of the light-induced oxygen release from spinach leaves which was then significantly suppressed in the presence of urea-type herbicide 3-(3,4-dichlorophenyl)−1,1-dimethylurea (DCMU). Our results clearly suggest that light-induced oxygen evolution can be monitored using the chip and suggesting that the Bio-LSI is a promising tool for real-time imaging. To the best of our knowledge, this report is the first to describe electrochemical imaging of light-induced O 2 evolution using LSI-based amperometric sensors in plants.

Tocopherols, lipid-soluble antioxidants play a crucial role in the antioxidant defense system in higher plants. The antioxidant function of α-tocopherol has been widely studied; however, experimental data on the formation of its oxidation products is missing. In this study, we attempt to provide spectroscopic evidence on the detection of oxidation products of α-tocopherol formed by its interaction with singlet oxygen and lipid peroxyl radical. Singlet oxygen was formed using photosensitizer rose bengal and thylakoid membranes isolated from Arabidopsis thaliana. Singlet oxygen reacts with polyunsaturated fatty acid forming lipid hydroperoxide which is oxidized by ferric iron to lipid peroxyl radical. The addition of singlet oxygen to double bond carbon on the chromanol head of α-tocopherol forms α-tocopherol hydroperoxide detected using fluorescent probe swallow-tailed perylene derivative. The decomposition of α-tocopherol hydroperoxide forms α-tocopherol quinone. The hydrogen abstraction from α-tocopherol by lipid peroxyl radical forms α-tocopheroxyl radical detected by electron paramagnetic resonance. Quantification of lipid and protein hydroperoxide from the wild type and tocopherol deficient (vte1) mutant Arabidopsis leaves using a colorimetric ferrous oxidation-xylenol orange assay reveals that α-tocopherol prevents formation of both lipid and protein hydroperoxides at high light. Identification of oxidation products of α-tocopherol might contribute to a better understanding of the protective role of α-tocopherol in the prevention of oxidative damage in higher plants at high light.

Oxidative modification of proteins in photosystem II (PSII) exposed to high light has been studied for a few decades, but the characterization of protein radicals formed by protein oxidation is largely unknown. Protein oxidation is induced by the direct reaction of proteins with reactive oxygen species known to form highly reactive protein radicals comprising carbon-centered (alkyl) and oxygen-centered (peroxyl and alkoxyl) radicals. In this study, protein radicals were monitored in Arabidopsis exposed to high light by immuno-spin trapping technique based on the detection of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) nitrone adducts using the anti-DMPO antibody. Protein radicals were imaged in Arabidopsis leaves and chloroplasts by confocal laser scanning microscopy using fluorescein conjugated with the anti-DMPO antibody. Characterization of protein radicals by standard blotting techniques using PSII protein specific antibodies shows that protein radicals are formed on D1, D2, CP43, CP47, and Lhcb3 proteins. Protein oxidation reflected by the appearance/disappearance of the protein bands reveals that formation of protein radicals was associated with protein fragmentation (cleavage of the D1 peptide bonds) and aggregation (cross-linking with another PSII subunits). Characterization of protein radical formation is important for better understating of the mechanism of oxidative modification of PSII proteins under high light.