No abstract

It is well known that biological systems, such as microorganisms, plants, and animals, including human beings, form spontaneous electronically excited species through oxidative metabolic processes. Though the mechanism responsible for the formation of electronically excited species is still not clearly understood, several lines of evidence suggest that reactive oxygen species (ROS) are involved in the formation of electronically excited species. This review attempts to describe the role of ROS in the formation of electronically excited species during oxidative metabolic processes. Briefly, the oxidation of biomolecules, such as lipids, proteins, and nucleic acids by ROS initiates a cascade of reactions that leads to the formation of triplet excited carbonyls formed by the decomposition of cyclic (1,2-dioxetane) and linear (tetroxide) high-energy intermediates. When chromophores are in proximity to triplet excited carbonyls, the triplet-singlet and triplet-triplet energy transfers from triplet excited carbonyls to chromophores result in the formation of singlet and triplet excited chromophores, respectively. Alternatively, when molecular oxygen is present, the triplet-singlet energy transfer from triplet excited carbonyls to molecular oxygen initiates the formation of singlet oxygen. Understanding the mechanism of the formation of electronically excited species allows us to use electronically excited species as a marker for oxidative metabolic processes in cells.

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 (O 2 •−) and singlet oxygen (1 O 2)] 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.

The exposure of human cells to oxidative stress leads to the oxidation of biomolecules such as lipids, proteins and nuclei acids. In this study, the oxidation of lipids, proteins and DNA was studied after the addition of hydrogen peroxide and Fenton reagent to cell suspension containing human leukemic monocyte lymphoma cell line U937. EPR spin-trapping data showed that the addition of hydrogen peroxide to the cell suspension formed hydroxyl radical via Fenton reaction mediated by endogenous metals. The malondialdehyde HPLC analysis showed no lipid peroxidation after the addition of hydrogen peroxide, whereas the Fenton reagent caused significant lipid peroxidation. The formation of protein carbonyls monitored by dot blot immunoassay and the DNA fragmentation measured by comet assay occurred after the addition of both hydrogen peroxide and Fenton reagent. Oxidative damage of biomolecules leads to the formation of singlet oxygen as conformed by EPR spin-trapping spectroscopy and the green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. It is proposed here that singlet oxygen is formed by the decomposition of high-energy intermediates such as dioxetane or tetroxide formed by oxidative damage of biomolecules.

Prenylquinols (tocochromanols and plastoquinols) serve as efficient physical and chemical quenchers of singlet oxygen ( O ) formed during high light stress in higher plants. Although quenching of O by prenylquinols has been previously studied, direct evidence for chemical quenching of O by plastoquinols and their oxidation products is limited in vivo. In the present study, the role of plastoquinol-9 (PQH -9) in chemical quenching of O was studied in Arabidopsis thaliana lines overexpressing the SOLANESYL DIPHOSPHATE SYNTHASE 1 gene (SPS1oex) involved in PQH -9 and plastochromanol-8 biosynthesis. In this work, direct evidence for chemical quenching of O by plastoquinols and their oxidation products is presented, which is obtained by microscopic techniques in vivo. Chemical quenching of O was associated with consumption of PQH -9 and formation of its various oxidized forms. Oxidation of PQH -9 by O leads to plastoquinone-9 (PQ-9), which is subsequently oxidized to hydroxyplastoquinone-9 [PQ(OH)-9]. We provide here evidence that oxidation of PQ(OH)-9 by O results in the formation of trihydroxyplastoquinone-9 [PQ(OH) -9]. It is concluded here that PQH -9 serves as an efficient O chemical quencher in Arabidopsis, and PQ(OH) -9 can be considered as a natural product of O reaction with PQ(OH)-9. The understanding of the mechanisms underlying O chemical quenching provides information on the role of plastoquinols and their oxidation products in the response of plants to photooxidative stress.

In this study, evidence is provided on the formation of electronically excited species in human multiple myeloma cells U266 in the growth medium exposed to hydrogen peroxide (H2O2). Two-dimensional imaging of ultra-weak photon emission using highly sensitive charge coupled device camera revealed that the addition of H2O2 to cell suspension caused the formation of triplet excited carbonyls 3(R = O)*. The kinetics of 3(R = O)* formation in the real time, as measured by one-dimensional ultra-weak photon emission using low-noise photomultiplier, showed immediate enhancement followed by a slow decay. In parallel to the formation of 3(R = O)*, the formation of singlet oxygen (1O2) in U266 cells caused by the addition of H2O2 was visualized by the imaging of 1O2 using the green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. Additionally, the formation of 1O2 after the addition of H2O2 to cell suspension was detected by electron paramagnetic resonance spin-trapping spectroscopy using 2,2,6,6-tetramethyl-4-piperidone. Presented results indicate that the addition of H2O2 to cell suspension results in the formation of 3(R = O)* and 1O2 in U266 cell suspension. The contribution of the cell-free medium to the formation of electronically excited species was discussed.

The U937 cell culture is a pro-monocytic, human histiocytic lymphoma cell line. These monocytes can differentiate into either macrophages or dendritic cells (antigenpresenting cells) depending on the initiators. The U937 cells activated in the presence of phorbol 12-myristate 13-acetate (PMA) change their morphology into macrophagelike cells creating pseudopodia and adhering generously. Macrophages are known to produce reactive oxygen species (ROS) mostly during phagocytosis of foreign particles, an important non-specific immune response. Recently, we have focused on the role of hydroxyl radical (HO •) and provide evidence on its importance for differentiation in U937 cells. Based on electron paramagnetic resonance (EPR) spectroscopy combined with confocal laser scanning microscopy (CLSM), formation of HO • was confirmed within the cells undergoing differentiation and/or apoptosis during the PMA treatment. This study aims to increase our knowledge of ROS metabolism in model cell lines used in human research.

Leaf senescence, accompanied by chlorophyll breakdown, chloroplast degradation and inhibition of photosynthesis, can be suppressed by an exogenous application of cytokinins. Two aromatic cytokinin arabinosides (6-benzylamino-9-β-d-arabinofuranosylpurines; BAPAs), 3-hydroxy- (3OHBAPA) and 3-methoxy- (3MeOBAPA) derivatives, have recently been found to possess high anti-senescence activity. Interestingly, their effect on the maintenance of chlorophyll content and maximal quantum yield of photosystem II (PSII) in detached dark-adapted leaves differed quantitatively in wheat (Triticum aestivum L. cv. Aranka) and Arabidopsis (Arabidopsisthaliana L. (Col-0)). In this work, we have found that the anti-senescence effects of 3OHBAPA and 3MeOBAPA in wheat and Arabidopsis also differ in other parameters, including the maintenance of carotenoid content and chloroplasts, rate of reduction of primary electron acceptor of PSII (QA) as well as electron transport behind QA, and partitioning of absorbed light energy in light-adapted leaves. In wheat, 3OHBAPA had a higher protective effect than 3MeOBAPA, whereas in Arabidopsis, 3MeOBAPA was the more efficient derivative. We have found that the different anti-senescent activity of 3OHBAPA and 3MeOBAPA was coupled to different ethylene production in the treated leaves: the lower the ethylene production, the higher the anti-senescence activity. 3OHBAPA and 3MeOBAPA also efficiently protected the senescing leaves of wheat and Arabidopsis against oxidative damage induced by both H2O2 and high-light treatment, which could also be connected with the low level of ethylene production.