Ultraviolet-B radiation applied to detached peach fruit: A study of free radical generation by EPR spin trapping.

In peaches, phenolic compounds are the major sources of antioxidants, and cyanidin-3-O-glucoside is the main anthocyanin present, above all in the skin. Anthocyanin content has been shown to increase after UV-B irradiation, which may be very harmful for all biological organisms due to the induction of the generation of reactive oxygen species (ROS). Peach fruits (cv. 'Suncrest') were exposed during post-harvest to supplemental ultraviolet-B radiation. A spin-trapping technique was used to monitor the generation of free radicals under UV-B, and 5-(diethoxy-phosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) was used as the spin trap. The flesh of peaches was essentially unaffected by the treatment, whereas the skin was responsive at the end of the treatment, accumulating ascorbate, flavonoids, cyanidin-3-O-glucoside, and showing a higher antioxidant activity. The levels of stable free radicals were also lower at the end of treatment. Carbon-centred radicals contributed the most to the total amounts of free radicals, whereas hydroxyl radicals and oxygen-centred free radicals contributed minimally. The carbon-centred free radical identified was the same as the one obtained after irradiation of authentic cyanidin-3-O-glucoside. During UV-B treatment cyanidin-3-O-glucoside increased and was capable of radicalization protecting the other organic molecules of the cell from oxidation. ROS, among which hydroxyl radicals, were thus maintained to minimal levels. This ability of cyanidin-3-O-glucoside displayed the mechanism underlined the tolerance to UV-B irradiation indicating that shelf life can be prolonged by the presence of anthocyanins. Thus, UV-B technique results a good approach to induce antioxidant production in peach fruits increasing their nutraceutical properties.

Antioxidative responses of Ocimum basilicum to sodium chloride or sodium sulphate salinization.

Soils and ground water in nature are dominated by chloride and sulphate salts. There have been several studies concerning NaCl salinity, however, little is known about the Na(2)SO(4) one. The effects on antioxidative activities of chloride or sodium sulphate in terms of the same Na(+) equivalents (25 mM Na(2)SO(4) and 50 mM NaCl) were studied on 30 day-old plants of Ocimum basilicum L., variety Genovese subjected to 15 and 30 days of treatment. Growth, thiobarbituric acid reactive substances (TBARS), relative ion leakage ratio (RLR), hydrogen peroxide (H(2)O(2)), ascorbate and glutathione contents as well as the activities of ascorbate peroxidase (APX, EC 1.11.1.11); glutathione reductase (GR, EC 1.6.4.2) and peroxidases (POD, EC 1.11.1.7) were determined. In leaves, growth was more depressed by 25 mM Na(2)SO(4) than 50 mM NaCl. The higher sensitivity of basil to Na(2)SO(4) was associated with an enhanced accumulation of H(2)O(2), an inhibition of APX, GR and POD activities (with the exception of POD under the 30-day-treatment) and a lower regeneration of reduced ascorbate (AsA) and reduced glutathione (GSH). However, the changes in the antioxidant metabolism were enough to limit oxidative damage, explaining the fact that RLR and TBARS levels were unchanged under both Na(2)SO(4) and NaCl treatment. Moreover, for both salts the 30-day-treatment reduced H(2)O(2) accumulation, unchanged RLR and TBARS levels, and enhanced the levels of antioxidants and antioxidative enzymes, thus achieving an adaptation mechanism against reactive oxygen species.

The influence of diluted seawater and ripening stage on the content of antioxidants in fruits of different tomato genotypes.

The aim of this study was to investigate if the combined effect of diluted seawater and ripening can improve the beneficial nutritional properties of tomato fruits from an antioxidant point of view. To reach the goal, different tomato cultivars and breeding lines, genetically modified for ripening, were investigated, and analysis of NADPH and NADP+ as well as of the main antioxidants such as ascorbic acid, lipoic acid, and tocopherols was performed at two ripening stages. The research was conducted on berries of the following genotypes of tomato: cv. Jama, Gimar wild type, Gimar gf, and Gimar nor. The mutant gf is a typical "stay green" mutant, characterized by an incomplete loss of chlorophyll; the nor mutation is characterized by a reduced biosynthesis of ethylene and carotenoids. Both ripening and salinity induced an oxidative stress, and the sensitivity to salt treatment was genotype-dependent. The genotypes cv. Jama and Gimar gf line showed increases in ascorbic acid, lipoic acid, and alpha-tocopherol during both ripening and salt treatment whereas total ascorbate and tocopherols decreased in the berries from salt-treated plants of Gimar wild type. Ripening also determined decreases in ascorbate and tocopherol amounts in the Gimar nor line where a positive effect of ripening and salinity was observed.

Enzymatic and non-enzymatic protective mechanisms in recalcitrant seeds of Araucaria bidwillii subjected to desiccation.

The changes in several antioxidants as well as in the level of C-centered free radicals and thiobarbituric acid reactive substances (TBARS) were studied in seeds of Araucaria bidwillii Hook desiccated to 37%, 28% and 21% moisture content. The lowest-safe moisture content for the seedling establishment was 37%. The embryo, besides double amounts of free radicals, showed higher levels of both enzymatic and non-enzymatic antioxidants than endosperm. Lutein decreased in both organs whereas alpha-tocopherol values were not affected by desiccation. In the embryo at 37% seed moisture content the antioxidant defense system increased giving rise to a decrease in free radicals. Beyond this point, free radicals and TBARS increased in agreement with the umpiring of the ascorbate/glutathione cycle by the decrease in reduced glutathione and glutathione reductase activity (GR, EC 1.6.4.2). At 21% moisture GR decreased. In the endosperm during desiccation, the consumption of ascorbate, total glutathione and lutein prevented the rise in free radicals and TBARS till 28% moisture, at which an increase in oxidized glutathione was also observed.

Photosystem II photochemical efficiency, zeaxanthin and antioxidant contents in the poikilohydric Ramonda serbica during dehydration and rehydration.

Changes in photochemical efficiency, non-radiative energy dissipation (NRD), de-epoxidation state of xanthophyll cycle components (DPS) and contents of the antioxidants ascorbic acid and glutathione were studied in leaves of the poikilohydric Ramonda serbica Panc. (Gesneriaceae) during cycles of dehydration and subsequent rehydration. In drying leaves, the intrinsic efficiency of PS II photochemistry and the photon yield of PS II electron transport showed strong progressive decreases. Simultaneously, the fraction of excitation energy dissipated as heat in the PS II antenna increased markedly. The energy-dependent component of non-photochemical quenching (NPQ) showed an increase in dehydrating leaves down to relative water contents (RWC) values near 30%. Further decreases in RWC below these values caused a decrease in NPQ. Accordingly, DPS showed a similar behaviour, with a sharp increase and a subsequent decrease at very low RWC, although the maximum DPS was reached at slightly lower RWC than that for the maximum NPQ. The pools of reduced ascorbate and glutathione increased strongly when the RWC values fell below 40% and remained high in fully dehydrated leaves. When plants were re-watered photosynthetic efficiency, NRD, DPS and antioxidant contents recovered their initial control values. However, during rehydration, the zeaxanthin content showed a transient increase, as did NPQ, indicating an increasing demand for non-radiative dissipation. On the other hand, the contents of reduced ascorbate and reduced glutathione decreased but were still relatively high in the initial phase of rehydration, when the rate of photosynthetic electron transport, proton pumping and NRD were still relatively low. These results indicate that several photoprotective mechanisms are operating in R. serbica. Protection from photo-oxidation and photoinhibition appears to be achieved by co-ordinated contributions by ascorbate, glutathione and zeaxanthin-mediated NPQ. This variety of photoprotective mechanisms may be essential for conferring desiccation-tolerance.

Protein dynamics in thylakoids of the desiccation-tolerant plant Boea hygroscopica during dehydration and rehydration.

Plants of Boea hygroscopica F. Muell were dehydrated to 9% relative water content (RWC) by withholding water for 26 d, and afterward the plants were rehydrated. Leaves were taken from control plants after 7, 12, and 26 d from the beginning of dehydration, and after 6 and 48 h from rehydration. The RWC decreased by 80% during dehydration, but the leaves regained RWC with rehydration. Dehydrated plants showed lesser amounts of proteins, lipids, and chlorophyll, all of which increased following rewatering. The lipid-to-protein ratio, which decreased during dehydration, returned to control level after 48 h of rehydration. Thylakoid lipids were more unsaturated when RWC reached the value of 9%. EPR measurements of spin-labeled proteins showed the presence of three different groups of proteins with different mobility in thylakoid membranes. The rotational correlation time of groups 1 and 2 increased with dehydration and decreased upon rehydration, whereas group 3 showed little changes. Desiccation did not cause thylakoid swelling or breakage, but the membrane system assemblage showed changes in thylakoid stacking. After 48 h of rehydration the membrane system recovered completely the organization of the fully hydrated state, showing several well-defined and regularly distributed grana.

Superoxide and hydroxyl radical generation, and superoxide dismutase in PSII membrane fragments from wheat.

Illumination of photosystem II (PSII) membrane fragments of wheat (Triticum durum Desf. cv. Adamello) gave rise to both O2*- and *OH radicals adducts of the novel spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO). With time, *OH became predominant displaying the conversion of O2*- into *OH. An intrinsic activity of superoxide dismutase (SOD) was found in PSII. Photoreduction of nitroblue tetrazolium (NBT) by PSII membrane fragments was induced by the addition of sodium azide and hydrogen peroxide. Western blotting of PSII proteins showed that a 29 kDa protein was recognised by an antibody against chloroplastic Fe-SOD from water lily. An increased formation rate of O2*- was observed in damaged PSII where the SOD activity decreased following a treatment with a free radical-generating system. Damage in PSII consisted also in a decrease in chlorophyll and in carotenoids as well as in a change in the lipid : chlorophyll : protein ratio. Upon treatment a decrease in the unsaturation of PSII membrane fragments was also monitored together with a degradation towards more saturated molecular species of monogalactosyldiacylglycerol.

Activated oxygen generation from thylakoids: a novel spin trap.

Using a novel phosphorylated spin trap, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), an analogue of the commonly used spin trap 5,5'-dimethyl-1-pyrroline N-oxide (DMPO), we have investigated the production of oxygen radical species under illumination of thylakoids from wheat (Triticum durum Desf cv. Ofanto). DEPMPO reacted with superoxide (O2*-) and hydroxyl radical (HO*) forming distinctive spin trap adducts. Spectra of (O2*-) and HO* adducts of DEPMPO were recorded in the presence of xanthine/xanthine oxidase and FeSO4/H2O2, respectively, and computer simulation of spectra was performed. During illumination of thylakoids both O2*- and HO* were detected as well. Transition metals catalysed transformation of O2*- into HO*. The conversion was enhanced by H2O2 and prevented by exogenous superoxide dismutase and catalase. The presence of a thylakoid-bound superoxide dismutase, whose activity was inhibited by H2O2 and diethyldithiocarbamic acid, was responsible for H2O2 production from O2*- and thus for HO* generation.