Technologies based on pulsed electric field (PEF) are increasingly pervasive in medical and industrial applications. However, the detailed understanding of how PEF acts on biosamples including proteins at the molecular level is missing. There are indications that PEF might act on biomolecules via electrogenerated reactive oxygen species (ROS). However, it is unclear how this action is modulated by the pro- and antioxidants, which are naturally present components of biosamples. This knowledge gap is often due to insufficient sensitivity of the conventionally utilized detection assays. To overcome this limitation, here we employed an endogenous (bio)chemiluminescence sensing platform, which enables sensitive detection of PEF-generated ROS and oxidative processes in proteins, to inspect effects of pro-and antioxidants. Taking bovine serum albumin (BSA) as a model protein, we found that the chemiluminescence signal arising from its solution is greatly enhanced in the presence of H 2 O 2 as a prooxidant, especially during PEF treatment. In contrast, the chemiluminescence signal decreases in the presence of antioxidant enzymes (catalase, superoxide dismutase), indicating the involvement of both H 2 O 2 and electrogenerated superoxide anion in oxidation-reporting chemiluminescence signal before, during, and after PEF treatment. We also performed additional biochemical and biophysical assays, which confirmed that BSA underwent structural changes after H 2 O 2 treatment, with PEF having only a minor effect. We proposed a scheme describing the reactions leading from interfacial charge transfer at the anode by which ROS are generated to the actual photon emission. Results of our work help to elucidate the mechanisms of action of PEF on proteins via electrogenerated reactive oxygen species and open up new avenues for the application of PEF technology. The developed chemiluminescence technique enables label-free, in-situ and non-destructive sensing of interactions between ROS and proteins. The technique may be applied to study oxidative damage of other classes of biomolecules such as lipids, nucleic acids or carbohydrates.

• MeSH
• Antioxidants * metabolism   MeSH
• Electricity MeSH
• Catalase metabolism   MeSH
• Luminescence MeSH
• Luminescent Measurements * methods   MeSH
• Oxidation-Reduction * MeSH
• Hydrogen Peroxide metabolism   MeSH
• Reactive Oxygen Species * metabolism   MeSH
• Serum Albumin, Bovine * metabolism   MeSH
• Cattle MeSH
• Animals MeSH
• Check Tag
• Cattle MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antioxidants * MeSH
• Catalase MeSH
• Hydrogen Peroxide MeSH
• Reactive Oxygen Species * MeSH
• Serum Albumin, Bovine * MeSH

Reactive oxygen species (ROS) are formed in photosystem II (PSII) under various types of abiotic and biotic stresses. It is considered that ROS play a role in chloroplast-to-nucleus retrograde signaling, which changes the nuclear gene expression. However, as ROS lifetime and diffusion are restricted due to the high reactivity towards biomolecules (lipids, pigments, and proteins) and the spatial specificity of signal transduction is low, it is not entirely clear how ROS might transduce signal from the chloroplasts to the nucleus. Biomolecule oxidation was formerly connected solely with damage; nevertheless, the evidence appears that oxidatively modified lipids and pigments are be involved in chloroplast-to-nucleus retrograde signaling due to their long diffusion distance. Moreover, oxidatively modified proteins show high spatial specificity; however, their role in signal transduction from chloroplasts to the nucleus has not been proven yet. The review attempts to summarize and evaluate the evidence for the involvement of ROS in oxidative signaling in PSII.

• Keywords
• Chloroplast-to-nucleus retrograde signaling, Lipid peroxidation, Protein oxidation, Reactive oxygen species,
• MeSH
• Chloroplasts * metabolism   MeSH
• Photosystem II Protein Complex * metabolism   MeSH
• Lipids MeSH
• Oxidative Stress MeSH
• Reactive Oxygen Species metabolism   MeSH
• Signal Transduction physiology   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Photosystem II Protein Complex * MeSH
• Lipids MeSH
• Reactive Oxygen Species MeSH

Mechanical injury or wounding in plants can be attributed to abiotic or/and biotic causes. Subsequent defense responses are either local, i.e. within or in the close vicinity of affected tissue, or systemic, i.e. at distant plant organs. Stress stimuli activate a plethora of early and late reactions, from electric signals induced within seconds upon injury, oxidative burst within minutes, and slightly slower changes in hormone levels or expression of defense-related genes, to later cell wall reinforcement by polysaccharides deposition, or accumulation of proteinase inhibitors and hydrolytic enzymes. In the current study, we focused on the production of reactive oxygen species (ROS) in wounded Arabidopsis leaves. Based on fluorescence imaging, we provide experimental evidence that ROS [superoxide anion radical (O2 •-) and singlet oxygen (1O2)] are produced following wounding. As a consequence, oxidation of biomolecules is induced, predominantly of polyunsaturated fatty acid, which leads to the formation of reactive intermediate products and electronically excited species.

• Keywords
• Arabidopsis, confocal microscopy, fluorescent probes, mechanical injury, wounding,
• Publication type
• Journal Article MeSH