Abstract
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.
Highlights
Plants are prone to various environmental stresses throughout their life cycle as high light, UV irradiation, heat, cold, drought and salinity (Murata et al, 2007; Choudhury et al, 2017)
We provided a characterization of protein radicals in the thylakoid membranes exposed to high light
Our results show the formation of protein radicals on two fragments at approximately 18 and 23 kDa and three aggregates at approximately 41, 55 and 68 kDa (Figure 5)
Summary
Plants are prone to various environmental stresses throughout their life cycle as high light, UV irradiation, heat, cold, drought and salinity (Murata et al, 2007; Choudhury et al, 2017). Plant responses to various types of stress factor by the formation of reactive oxygen species (ROS) are known to play a crucial role in the retrograde signaling and oxidative damage (Schmitt et al, 2014; Laloi and Havaux, 2015; Dietz et al, 2016; Mittler, 2017). Protein Radicals in Plants in the PSII antenna (CP43, CP47, and Lhcb proteins) and PSII reaction center (D1, D2) proteins. Hydroxyl radical (HO) is formed by metal-mediated reduction of hydrogen peroxide at the site of metal coordination to PSII reaction center proteins (Pospíšil, 2014). The oxidation of protein by ROS forms carboncentered (alkyl) and oxygen-centered (peroxyl and alkoxyl) protein radicals (Davies, 2016). It was predominantly due to a limitation in the detection of protein radicals caused by high reactivity of protein radicals toward other proteins and short lifetime of protein radicals (Mattila et al, 2015)
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