Abstract
In the current study, singlet oxygen formation by lipid peroxidation induced by heat stress (40 °C) was studied in vivo in unicellular green alga Chlamydomonas reinhardtii. Primary and secondary oxidation products of lipid peroxidation, hydroperoxide and malondialdehyde, were generated under heat stress as detected using swallow-tailed perylene derivative fluorescence monitored by confocal laser scanning microscopy and high performance liquid chromatography, respectively. Lipid peroxidation was initiated by enzymatic reaction as inhibition of lipoxygenase by catechol and caffeic acid prevented hydroperoxide formation. Ultra-weak photon emission showed formation of electronically excited species such as triplet excited carbonyl, which, upon transfer of excitation energy, leads to the formation of either singlet excited chlorophyll or singlet oxygen. Alternatively, singlet oxygen is formed by direct decomposition of hydroperoxide via Russell mechanisms. Formation of singlet oxygen was evidenced by the nitroxyl radical 2,2,6,6-tetramethylpiperidine-1-oxyl detected by electron paramagnetic resonance spin-trapping spectroscopy and the imaging of green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. Suppression of singlet oxygen formation by lipoxygenase inhibitors indicates that singlet oxygen may be formed via enzymatic lipid peroxidation initiated by lipoxygenase.
Highlights
Reactive oxygen species (ROS) are formed by activation of non-reactive molecular oxygen during photosynthetic light reaction in chloroplasts, cellular respiration in mitochondria and defence against microorganisms in phagocyte plasma membrane[1]
The integral distribution of Spy-LHPOx fluorescence intensity shows that Spy-LHPOx fluorescence in heated Chlamydomonas cells is enhanced by about 4 times as compared to non-heated Chlamydomonas cells. These results reveal that exposure of Chlamydomonas cells to heat stress leads to the formation of LOOH
In agreement with this evidence, we showed that the exposure of Chlamydomonas cells to 40 °C leads to the formation of LOOH (Fig. 1)
Summary
Reactive oxygen species (ROS) are formed by activation of non-reactive molecular oxygen during photosynthetic light reaction in chloroplasts, cellular respiration in mitochondria and defence against microorganisms in phagocyte plasma membrane[1]. Source of electronically excited species in biological systems[10,11,12,13,14] In this reaction, LOOH is oxidized to LOO under oxidizing conditions such are oxidized transition metals, ferric heme iron of cytochrome c, peroxynitrite, chloroperoxide, and hypochlorous acid. Peroxyl radical might either undergoes cyclization to dioxetane or recombines to tetroxide[11,15,16,17,18,19] These high energy intermediates decompose to triplet excited carbonyls (3L = O*) which might transfer triplet energy either to pigments forming excited pigments or molecular oxygen forming 1O210,20–24. In agreement with this proposal, experimental data from many models indicate that the exposure of photosynthetic organisms to heat stress leads to the formation of lipid peroxidation secondary product, malondialdehyde (MDA), as detected by TBARS assay[31,32,33,34]. It has been demonstrated that exposure of leaf and root segments of Phalaenopsis to 40 °C enhanced lipoxygenase activity and MDA formation[31,32,33,34]
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