New prenyllipid metabolites identified in Arabidopsis during photo‐oxidative stress

In the present study, we have identified new prenyllipid metabolites formed during high light stress in Arabidopsis thaliana, whose origin and function remained unknown so far. It was found that plastoquinone‐C accumulates mainly in the reduced form under high light conditions, as well as during short‐term excess light illumination both in the wild‐type and tocopherol biosynthetic vte1 mutant, suggesting that plastoquinone‐C, a singlet oxygen‐derived prenyllipid, is reduced in chloroplasts by photosystem II or enzymatically, outside thylakoids. Plastoquinone‐B, a fatty acid ester of plastoquinone‐C, was identified for the first time in Arabidopsis in high light grown wild‐type plants and during short‐time, excess light illumination of the wild‐type plants and the vte1 mutant. The gene expression analysis showed that vte2 gene is most pronouncedly up‐regulated among the prenyllipid biosynthetic genes under high light and induction of its expression is mainly caused by an increased level of singlet oxygen, as was demonstrated in experiments with D₂O‐treated plants under excess light conditions.     

Sodium azide as a specific quencher of singlet oxygen during chemiluminescent detection by luminol and Cypridina luciferin analogues

Reactive oxygen species (ROS) are presently thought to play important role in an increasing number of the physiological and pathological processes in living organisms. Various chemiluminescent (CL) compounds have been studied in order to find suitable and specific probes for the detection of particular ROS species. The CL of luminol is known to be non‐specific and can be induced by various oxidants. Two Cypridina luciferin analogues, CLA and MCLA, have been used for the detection of ROS in vivo. CLAs are thought to emit light only when reacting with superoxide and singlet oxygen. It is possible to distinguish the particular ROS by using a specific quencher or scavenger, e.g. superoxide dismutase (SOD) or sodium azide (NaN₃). The CL reactions of luminol (3‐aminophthalhydrazide), CLA [2‐methyl‐6‐phenyl‐3,7‐dihydroimidazo(1,2α) pyrazin‐3‐one] and MCLA [2‐methyl‐6‐(p‐methoxyphenyl)‐3,7‐dihydroimidazo(1,2α) pyrazin‐3‐one] were studied in three hydrogen peroxide decomposition systems (H₂O₂–HRP; H₂O₂–CuSO₄; and H₂O₂–NaOCl). The measurements were carried out in phosphate buffer, pH 7.4, at 25 °C, using a luminometer (Fluoroskan Ascent FL and Sirius C). NaN₃ was used as the specific quencher of singlet oxygen. The results demonstrate that the proclaimed specifity of the CL of Cypridina luciferin analogues towards singlet oxygen has to be discussed. Copyright © 2011 John Wiley & Sons, Ltd.     

Imaging of ultra‐weak bio‐chemiluminescence and singlet oxygen generation in germinating soybean in response to wounding

Ultra‐weak bio‐chemiluminescence (UBC) from germinating soybean (Glycine max L. Merr) cotyledon under mechanical wounding was observed using a high‐sensitivity imaging system based on an ICCD detector and a highly sensitive single photon counter (SPC) device. The UBC imaging showed that the intensity at the injury location on a wounded cotyledon was obviously enhanced as compared with that at the non‐injured point. The UBC intensity of wounded cotyledons was initially very high and reached a stationary state after about 5 min. Wounding‐induced emission could be suppressed by wounding in the presence of sodium azide. Deuterium oxide amplified the emission intensity. It was concluded that singlet oxygen (¹O₂) was the main cause of the emission during the wounding phase. Copyright © 2002 John Wiley & Sons, Ltd.     

Aerobic photoreactivity of synthetic eumelanins and pheomelanins: generation of singlet oxygen and superoxide anion

In this work, we examined photoreactivity of synthetic eumelanins, formed by autooxidation of DOPA, or enzymatic oxidation of 5,6‐dihydroxyindole‐2‐carboxylic acid and synthetic pheomelanins obtained by enzymatic oxidation of 5‐S‐cysteinyldopa or 1:1 mixture of DOPA and cysteine. Electron paramagnetic resonance oximetry and spin trapping were used to measure oxygen consumption and formation of superoxide anion induced by irradiation of melanin with blue light, and time‐resolved near‐infrared luminescence was employed to determine the photoformation of singlet oxygen between 300 and 600 nm. Both superoxide anion and singlet oxygen were photogenerated by the synthetic melanins albeit with different efficiency. At 450‐nm, quantum yield of singlet oxygen was very low (~10^?4) but it strongly increased in the UV region. The melanins quenched singlet oxygen efficiently, indicating that photogeneration and quenching of singlet oxygen may play an important role in aerobic photochemistry of melanin pigments and could contribute to their photodegradation and photoaging.     

The effects of detergents DDM and β-OG on the singlet excited state lifetime of the chlorophyll a in cytochrome b6f complex from spinach chloroplasts

The singlet excited state lifetime of the chlorophyll a (Chl a) in cytochrome b ₆ f (Cyt b ₆ f) complex was reported to be shorter than that of free Chl a in methanol, but the value was different for Cyt b ₆ f complexes from different sources (∼200 and ∼600 ps are the two measured results). The present study demonstrated that the singlet excited state lifetime is associated with the detergents n-dodecyl-β-D-maltoside (DDM) and n-octyl-β-D-glucopyranoside (β-OG), but has nothing to do with the different sources of Cyt b ₆ f complexes. Compared with the Cyt b ₆ f dissolved in β-OG, the Cyt b ₆ f in DDM had a lower fluorescence yield, a lower photodegradation rate of Chl a, and a shorter lifetime of Chl a excited state. In short, the singlet excited state lifetime, ∼200 ps, of the Chl a in Cyt b ₆ f complex in DDM is closer to the true in vivo.     

Photoinhibition of carotenoidless reaction centers from Rhodobacter sphaeroides by visible light. Effects on protein structure and electron transport

Inhibition of electron transport and damage to the protein subunits by visible light has been studied in isolated reaction centers of the non-sulfur purple bacterium Rhodobacter sphaeroides. Illumination by 1100 μEm⁻² s⁻¹ light induced only a slight effect in wild type, carotenoid containing 2.4.1. reaction centers. In contrast, illumination of reaction centers isolated from the carotenoidless R26 strain resulted in the inhibition of charge separation as detected by the loss of the initial amplitude of absorbance change at 430 nm arising from the P⁺Q_B ⁻ → PQ_B recombination. In addition to this effect, the L, M and H protein subunits of the R26 reaction center were damaged as shown by their loss on Coomassie stained gels, which was however not accompanied by specific degradation products. Both the loss of photochemical activity and of protein subunits were suppressed in the absence of oxygen. By applying EPR spin trapping with 2,2,6,6-tetramethylpiperidine we could detect light-induced generation of singlet oxygen in the R26, but not in the 2.4.1. reaction centers. Moreover, artificial generation of singlet oxygen, also led to the loss of the L, M and H subunits. Our results provide evidence for the common hypothesis that strong illumination by visible light damages the carotenoidless reaction center via formation of singlet oxygen. This mechanism most likely proceeds through the interaction of the triplet state of reaction center chlorophyll with the ground state triplet oxygen in a similar way as occurs in Photosystem II. This revised version was published online in August 2006 with corrections to the Cover Date.     

Photobleaching of pheomelanin increases its phototoxic potential: Physicochemical studies of synthetic pheomelanin subjected to aerobic photolysis

Although melanin is a photoprotective pigment, its elevated photochemical reactivity could lead to various phototoxic processes. Photoreactivity of synthetic pheomelanin, derived from 5‐S‐cysteinyldopa (5SCD‐M) and its photodegradation products obtained by subjecting the melanin to aerobic irradiation with UV‐visible light, was examined employing an array of advanced physicochemical methods. Extensive photolysis of 5SCD‐M was accompanied by partial bleaching of the melanin, modification of its paramagnetic properties, and significant increase in the ability to photogenerate singlet oxygen. The changes correlated with a substantial decrease in the melanin content of benzothiazine (BT) units and increase of modified benzothiazole (BZ) units. Synthetically prepared BZ exhibited higher efficiency to photogenerate singlet oxygen than the synthetic BT, and the free radical form of BZ, unlike that of BT, did not show measurable spin density on nitrogen atom, which was confirmed by quantum chemical calculations. Formation of modified BZ units in the photobleached 5SCD‐M is responsible for the paramagnetic and photochemical changes of the melanin and its elevated phototoxic potential. Given a relatively constant pheomelanin–eumelanin ratio, such undesirable changes could occur in individual of all skin types.     

Chemical quenching of singlet oxygen by plastoquinols and their oxidation products in Arabidopsis

Prenylquinols (tocochromanols and plastoquinols) serve as efficient physical and chemical quenchers of singlet oxygen (¹O₂) formed during high light stress in higher plants. Although quenching of ¹O₂ by prenylquinols has been previously studied, direct evidence for chemical quenching of ¹O₂ by plastoquinols and their oxidation products is limited in vivo. In the present study, the role of plastoquinol‐9 (PQH₂‐9) in chemical quenching of ¹O₂ was studied in Arabidopsis thaliana lines overexpressing the SOLANESYL DIPHOSPHATE SYNTHASE 1 gene (SPS1oex) involved in PQH₂‐9 and plastochromanol‐8 biosynthesis. In this work, direct evidence for chemical quenching of ¹O₂ by plastoquinols and their oxidation products is presented, which is obtained by microscopic techniques in vivo. Chemical quenching of ¹O₂ was associated with consumption of PQH₂‐9 and formation of its various oxidized forms. Oxidation of PQH₂‐9 by ¹O₂ 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 ¹O₂ results in the formation of trihydroxyplastoquinone‐9 [PQ(OH)₃‐9]. It is concluded here that PQH₂‐9 serves as an efficient ¹O₂ chemical quencher in Arabidopsis, and PQ(OH)₃‐9 can be considered as a natural product of ¹O₂ reaction with PQ(OH)‐9. The understanding of the mechanisms underlying ¹O₂ chemical quenching provides information on the role of plastoquinols and their oxidation products in the response of plants to photooxidative stress.     

Hydroxy‐plastochromanol and plastoquinone‐C as singlet oxygen products during photo‐oxidative stress in Arabidopsis

In the present study, we have shown that hydroxy‐plastochromanol and plastoquinone‐C, the hydroxy derivatives of plastochromanol and plastoquinone‐9, respectively, are specifically formed from the parent compounds upon action of singlet oxygen and can be regarded as stable, specific, natural products of singlet oxygen action during photo‐oxidative stress in vivo. The presented data indicate that plastoquinone‐C formation dominates mainly during relatively short periods of high light stress where efficient production of singlet oxygen takes place, whereas hydroxy‐plastochromanol is rather formed under conditions of long‐term, less pronounced generation of singlet oxygen. An interesting observation was that hydroxy‐plastochromanol is formed even at very low light conditions (5–10 μmol photons m^?2 s^?1), indicating that singlet oxygen is generated not only during high light stress but also its formation by photosystem II is inseparably connected with the functioning of this photosystem even at the lowest light intensities.     

Rapid determination of rice seed vigour by spontaneous chemiluminescence and singlet oxygen generation during early imbibition

Using a highly sensitive single photon counter, a spontaneous chemiluminescence (CL) study on rice (Oryza sativa L.) seeds stored in different years was carried out. We first observed that the degree of ageing in rice seeds was related to the intensity of spontaneous CL during early imbibition (0–30 min). Rice seeds stored for a shorter time had a stronger intensity of CL in early imbibition. The germination rate of rice seeds showed an obvious positive correlation with the intensity of spontaneous CL. Singlet oxygen (¹O₂) in rice seeds during early imbibition was investigated by a CL method using a cypridina luciferin analogue, 2‐methyl‐6‐(p‐methoxyphenyl)‐3,7‐dihydroimidazo [1,2α] pyrazin‐3‐one (MCLA), as a selective CL probe. Additional experimental evidence for the formation of ¹O₂ came from the quenching effect of sodium azide (NaN₃) on MCLA‐mediated rice seeds' CL. Analysis based on the experimental results demonstrated that spontaneous CL in rice seeds during early imbibition was mainly contributed by singlet oxygen (¹O₂). Copyright © 2002 John Wiley & Sons, Ltd.     

Liquid Crystallinity as a Self‐Assembly Motif for High‐Efficiency,Solution‐Processed,Solid‐State Singlet Fission Materials

Solution and solution‐deposited thin films of the discotic liquid crystalline electron acceptor–donor–acceptor (A‐D‐A) p‐type organic semiconductor FHBC(TDPP)₂, synthesized by coupling thienyl substituted diketopyrrolopyrrole (TDPP) onto a fluorenyl substituted hexa‐peri‐hexabenzocoronene (FHBC) core, are examined by ultrafast and nanosecond transient absorption spectroscopy, and time‐resolved photoluminescence studies to examine their ability to support singlet fission (SF). Grazing incidence wide‐angle X‐ray (GIWAX) studies indicate that as‐cast thin films of FHBC(TDPP)₂ are “amorphous,” while hexagonal packed discotic liquid crystalline films evolve during thermal annealing. SF in as‐cast thin films is observed with an ≈150% triplet generation yield. Thermally annealing the thin films improves SF yields up to 170%. The as‐cast thin films show no long‐range order, indicating a new class of SF material where the requirement for local order and strong near neighbor coupling has been removed. Generation of long‐lived triplets (µs) suggests that these materials may also be suitable for inclusion in organic solar cells to enhance performance.     

Investigation on 1O2 generation ability of di‐sulfonic phthalocyanine zinc isomers using an HPLC–CL system

An HPLC system combining a chemiluminescence detector was applied to estimate the singlet oxygen (¹O₂) generation ability of di‐sulfonic phthalocyanine zinc (ZnPcS₂) isomers. As photosensitizers, ZnPcS₂ produces ¹O₂ in air‐saturated solutions under photoirradiation. The latter reacts with methyl Cypridina luciferin analogue (MCLA) to initiate chemiluminescence. This photoinduced chemiluminescence (PCL) of MCLA provides an easy method for evaluating the isomers' ¹O₂ generation ability during a simultaneous HPLC separation procedure. The cis‐isomers and trans‐isomers of ZnPcS₂ show different ¹O₂ generation abilities, which are in accordance with differences in their absorption spectra. Copyright © 2013 John Wiley & Sons, Ltd.     

A compact fiber‐optic probe‐based singlet oxygen luminescence detection system

This paper presents a novel compact fiberoptic based singlet oxygen near‐infrared luminescence probe coupled to an InGaAs/InP single photon avalanche diode (SPAD) detector. Patterned time gating of the single‐photon detector is used to limit unwanted dark counts and eliminate the strong photosensitizer luminescence background. Singlet oxygen luminescence detection at 1270 nm is confirmed through spectral filtering and lifetime fitting for Rose Bengal in water, and Photofrin in methanol as model photosensitizers. The overall performance, measured by the signal‐to‐noise ratio, improves by a factor of 50 over a previous system that used a fiberoptic‐coupled superconducting nanowire single‐photon detector. The effect of adding light scattering to the photosensitizer is also examined as a first step towards applications in tissue in vivo.

     

Prooxidant action of carazolol in the Fenton‐like reaction

Carazolol [4‐(2‐hydroxy‐3‐isopropyl‐amino‐propoxy)‐carbazole], a β₃‐adrenoceptor agonist, is clinically used in the treatment of hypertension, cardiac arrhythmias and angina pectoris. Despite the beneficial effect of the drug, its high dose may contribute to cardiotoxicity. This study was conducted to examine whether carazolol can influence hydroxyl radical formation by a Fenton‐like reaction [Co(II) + H₂O₂ + HO^‐] in the presence of ethylenediaminetetraacetic acid. The oxygen free radicals and singlet oxygen (¹O₂) formation was traced by three different assay methods: chemiluminescence (CL), an electron spin resonance (ESR) spin trapping with 2,2,6,6‐tetramethyl‐4‐piperidine and 5,5‐dimethyl‐1‐pyrroline‐1‐oxide, and spectrophotometric determination of ¹O₂ based on bleaching of p‐nitrosodimethylaniline. The effect of hydroxyl radical inhibitors and ¹O₂ quenchers on peroxidation of carazolol was also examined. The results indicated that carazolol enhanced the HO radical and ¹O₂ formation in a Fenton‐like reaction. Copyright © 2010 John Wiley & Sons, Ltd.     

Singlet oxygen is part of a hyperoxidant state generated during spore germination

We show that singlet oxygen is generated in asexual spores (conidia) from Neurospora crassa at the onset of germination. Oxidation of N. crassa catalase-1 (Cat-1) was previously shown to be caused by singlet oxygen (Lledías et al. J. Biol. Chem. 273, 1998). In germinating conidia, increased protein oxidation, decrease of total protein, Cat-1 oxidation and accumulation of cat-1 mRNA was detected. These changes were modulated in vivo by light intensity, an external clean source of singlet oxygen, and by carotene amount and content of coordinated double bonds. Conditions that stimulated singlet oxygen formation increased Cat-1 oxidation and accumulation of cat-1 mRNA. Germinating conidia from mutant strains altered in carotene synthesis showed increased levels of protein degradation, Cat-1 oxidation and accumulation of cat-1 mRNA. During germination Cat-1a was oxidized, oxidized Cat-1c-Cat-1e conformers disappeared and Cat-1a was synthesized de novo. Furthermore, spontaneous oxygen-dependent chemiluminescence increased as soon as conidia absorbed dissolved oxygen. Low-level chemiluminescence is due to photon emission from excited electrons in carbonyls and singlet oxygen as they return to their ground state. H2O2 added to conidia under Ar caused a peak of chemiluminescence and germination of 20% of conidia, suggesting that a hyperoxidant state suffices to start germination under anaerobic conditions. Taken together, these results show that singlet oxygen is part of a hyperoxidant state that develops at the start of germination of conidia, in consonance with our proposal that morphogenetic transitions occur as a response to a hyperoxidant state.     

Chemiluminescence Emission during Reactions between Superoxide and Selected Aliphatic and Aromatic Halocarbons in Aprotic Media

The reactions between superoxide free radical anion (.O⁻₂) with the halocarbons CCl₄, CHCl₃, BrCH₂CH₂Br(EDB), decachloro-biphenyl (DCBP), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in dimethyl sulphoxide (DMSO) results in the emission of chemiluminescence (CL). The chemiluminescence reactions are characterized as having biphasic second order kinetics, CL wavelengths between 350 nm and 650 nm, and exhibiting perturbation by chemicals reactive with singlet oxygen. These data suggest that singlet oxygen species are the excited state responsible for the light emissions. Polarographic studies confirm .O⁻₂ consumption and halide release in the reactions, while gas liquid chromatography and NBT reduction demonstrate the decomposition of the halocarbons into products. A chemiluminescent reaction mechanism is proposed involving reductive dehalogenation of the halocarbons and the generation of singlet oxygen. The significance of singlet oxygen generation is discussed with respect to a general mechanism for explaining the rapid initiation of lipid peroxidative membrane damage in halocarbon toxigenicity in animal and plant tissues.     

Application of a sensitive near‐field microwave microprobe to the nondestructive characterization of microbial rhodopsin

We study the opto‐electrical properties of Natronomonas pharaonis sensory rhodopsin II (NpSRII) by using a near‐field microwave microprobe (NFMM) under external light illumination. To investigate the possibility of application of NFMM to biological macromolecules, we used time dependent properties of NPSRII before/after light activation which has three distinct states – ground‐state, M‐state, and O‐state. The diagnostic ability of NFMM is demonstrated by measuring the microwave reflection coefficient (S₁₁) spectrum of NpSRII under steady‐state illumination in the wavelength range of 350–650 nm. Moreover, we present microwave reflection coefficient S₁₁ spectra in the same wavelength range for two fast‐photocycling rhodopsins: green light‐absorbing proteorhodopsin (GPR) and Gloeobacter rhodopsin (GR). In addition the frequency sweep shift can be detected completely even for tiny amounts of sample (～10^–3 OD of rhodopsin). Based on these results NFMM shows both very high sensitivity for detecting conformational changes and produces a good time‐resolved spectrum. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Singlet oxygen scavenging activity of tocopherol and plastochromanol in Arabidopsis thaliana: relevance to photooxidative stress

In the present study, singlet oxygen (¹O₂) scavenging activity of tocopherol and plastochromanol was examined in tocopherol cyclase‐deficient mutant (vte1) of Arabidopsis thaliana lacking both tocopherol and plastochromanol. It is demonstrated here that suppression of tocopherol and plastochromanol synthesis in chloroplasts isolated from vte1 Arabidopsis plants enhanced ¹O₂ formation under high light illumination as monitored by electron paramagnetic resonance spin‐trapping spectroscopy. The exposure of vte1 Arabidopsis plants to high light resulted in the formation of secondary lipid peroxidation product malondialdehyde as determined by high‐pressure liquid chromatography. Furthermore, it is shown here that the imaging of ultra‐weak photon emission known to reflect oxidation of lipids was unambiguously higher in vte1 Arabidopsis plants. Our results indicate that tocopherol and plastochromanol act as efficient ¹O₂ scavengers and protect effectively lipids against photooxidative damage in Arabidopsis plants.     

The effects of detergents DDM and β-OG on the singlet excited state lifetime of the chlorophyll a in cytochrome b6f complex from spinach chloroplasts

The singlet excited state lifetime of the chlorophyll a (Chi a) in cytochrome b6f (Cyt b6f) complex was reported to be shorter than that of free Chl a in methanol, but the value was different for Cyt b6f complexes from different sources (～200 and ～600 ps are the two measured results). The present study demonstrated that the singiet excited state lifetime is associated with the detergents n-dodecyl-β-D-maltoside (DDM) and n-octyl-β-D-glucopyranoside (β-OG), but has nothing to do with the different sources of Cyt b6f complexes. Compared with the Cyt b6f dissolved in β-OG, the Cyt b6f in DDM had a lower fluorescence yield, a lower photodegradation rate of Chl a, and a shorter lifetime of Chl a excited state. In short, the singlet excited state lifetime, ～200 ps, of the Chl a in Cyt b6f complex in DDM is closer to the true in vivo.     

Evidence of singlet oxygen evolution by whole living cells of Chlamydomonas reinhardtii

The oxygen evolved by Chlamydomonas reinhardtii in the light is measured simultaneously with a Clark electrode and with the nitrosodimethylaniline-imidazole colorimetric method which is specific for singlet oxygen. Experiments with wild-type and FuD7 mutant cells (unable to synthesize the D1 protein of Photosystem II), with dichlorophenyldimethylurea (which blocks electron transfer from Photosystem II to Photosystem I) and with dibromothymoquinone (which diverts electrons from their normal path between the two photosystems), as well as with hydroxylamine (an inactivator of the water-splitting part of Photosystem II and a competitor of water for electron donation to it), all point to the dependence of detected singlet oxygen on photolysis of water by Photosystem II.Abbreviations DBMIB Dibromothymoquinone - DCMU Dichlorophenyldimethylurea - PS I and PS II Photosystems I and II - RNO para-nitrosodimethylaniline Contribution of the Centre interdisciplinaire de Biochimie de Oxygène.